bims-axbals Biomed News
on Axonal biology and ALS
Issue of 2025–02–02
fifty-nine papers selected by
TJ Krzystek, ALS Therapy Development Institute
  1. Sporadic ALS iPSC-derived motor neurons show axonal defects linked to altered axon guidance pathways.
  2. Endogenous LRRK2 and PINK1 function in a convergent neuroprotective ciliogenesis pathway in the brain.
  3. Rapid iPSC-derived neuromuscular junction model uncovers motor neuron dominance in amyotrophic lateral sclerosis cytopathy.
  4. Understanding retinal tau pathology through functional 2D and 3D iPSC-derived in vitro retinal models.
  5. Small Extracellular Vesicles Promote Axon Outgrowth by Engaging the Wnt-Planar Cell Polarity Pathway.
  6. NPT100-18A rescues mitochondrial oxidative stress and neuronal degeneration in human iPSC-based Parkinson's model.
  7. Tail Anchored protein insertion mediated by CAML and TRC40 links to neuromuscular function in mice.
  8. Modeling and correction of protein conformational disease in iPSC-derived neurons through personalized base editing.
  9. Prognostic Value of Cerebrospinal Fluid and Serum Neurofilament Light Chain in Amyotrophic Lateral Sclerosis: A Correlation Study.
  10. A role for mitochondria-ER crosstalk in amyotrophic lateral sclerosis 8 pathogenesis.
  11. Amyotrophic lateral sclerosis caused by FUS mutations: advances with broad implications.
  12. Intramuscular inhibition of glycogen phosphorylase improves motor function in spinal cord injury.
  13. A cellular model of TDP-43 induces phosphorylated TDP-43 aggregation with distinct changes in solubility and autophagy dysregulation.
  14. Advances in the Development and Application of Human Organoids: Techniques, Applications, and Future Perspectives.
  15. TDP-43 nuclear retention is antagonized by hypo-phosphorylation of its C-terminus in the cytoplasm.
  16. Clinical perspective on pluripotent stem cells derived cell therapies for the treatment of neurodegenerative diseases.
  17. Resting-State EEG Oscillations in Amyotrophic Lateral Sclerosis (ALS): Toward Mechanistic Insights and Clinical Markers.
  18. Systematic Evaluation of Extracellular Coating Matrix on the Differentiation of Human-Induced Pluripotent Stem Cells to Cortical Neurons.
  19. Cryo-ET suggests tubulin chaperones form a subset of microtubule lumenal particles with a role in maintaining neuronal microtubules.
  20. APP antisense oligonucleotides are effective in rescuing mitochondrial phenotypes in human iPSC-derived trisomy 21 astrocytes.
  21. Activation of lysophagy by a TBK1-SCFFBXO3-TMEM192-TAX1BP1 axis in response to lysosomal damage.
  22. Optimization of Transcription Factor-Driven Neuronal Differentiation from Human Induced Pluripotent Stem Cells for Disease Modelling and Drug Screening.
  23. Transforming amyotrophic lateral sclerosis into a liveable disease.
  24. Ferroptosis triggers mitochondrial fragmentation via Drp1 activation.
  25. Biotin mitigates the development of manganese-induced, Parkinson's disease-related neurotoxicity in Drosophila and human neurons.
  26. Mitochondria-plasma membrane contact sites regulate the ER-mitochondria encounter structure.
  27. Glucose Transporter 1 Deficiency Impairs Glucose Metabolism and Barrier Induction in Human Induced Pluripotent Stem Cell-Derived Astrocytes.
  28. VDAC1: A Key Player in the Mitochondrial Landscape of Neurodegeneration.
  29. Early-onset sleep alterations found in patients with amyotrophic lateral sclerosis are ameliorated by orexin antagonist in mouse models.
  30. Protocol for assessing myelination by human iPSC-derived oligodendrocytes in Shiverer mouse ex vivo brain slice cultures.
  31. Mutations in hnRNP A1 drive neurodegeneration and alternative RNA splicing of neuronal gene targets.
  32. Intraventricular Administration of Exosomes from Patients with Amyotrophic Lateral Sclerosis Provokes Motor Neuron Disease in Mice.
  33. The role of mitochondrial dysfunction in Huntington's disease: Implications for therapeutic targeting.
  34. Two- and Three-Dimensional In Vitro Models of Parkinson's and Alzheimer's Diseases: State-of-the-Art and Applications.
  35. Morphological Characteristics and Extracellular Matrix Abnormalities in Astrocytes Derived From iPSCs of Children With Alexander Disease.
  36. Novel Poly-Arginine Peptide R18D Reduces α-Synuclein Aggregation and Uptake of α-Synuclein Seeds in Cortical Neurons.
  37. Stem Cell Therapy for the Treatment of Amyotrophic Lateral Sclerosis: Comparison of the Efficacy of Mesenchymal Stem Cells, Neural Stem Cells, and Induced Pluripotent Stem Cells.
  38. Primary Neuronal Culture and Transient Transfection.
  39. Quantitative Analysis of Mitochondria-Associated Endoplasmic Reticulum Membrane (MAM) Stabilization in a Neural Model of Alzheimer's Disease (AD).
  40. Splicing accuracy varies across human introns, tissues, age and disease.
  41. The ubiquitin-conjugating enzyme UBE2D maintains a youthful proteome and ensures protein quality control during aging by sustaining proteasome activity.
  42. Super-resolution imaging of proteins inside live mammalian cells with mLIVE-PAINT.
  43. SLC25A38 is required for mitochondrial pyridoxal 5'-phosphate (PLP) accumulation.
  44. Oxyglutamate Carrier Alleviates Cerebral Ischaemia-Reperfusion Injury by Regulating Mitochondrial Function.
  45. Inflammatory signatures of microglia in hypercortisolemia-related depression.
  46. Roles of C/EBPβ/AEP in Neurodegenerative Diseases.
  47. Increased expression of the small lysosomal gene SVIP in the Drosophila gut suppresses pathophysiological features associated with a high-fat diet.
  48. The Type III Intermediate Filament Protein Peripherin Regulates Lysosomal Degradation Activity and Autophagy.
  49. Novel strategies targeting mitochondria-lysosome contact sites for the treatment of neurological diseases.
  50. Mitochondrial fission and fusion in neurodegenerative diseases:Ca2+ signalling.
  51. New Atg9 Phosphorylation Sites Regulate Autophagic Trafficking in Glia.
  52. Data-Driven Maturity Level Evaluation for Cardiomyocytes Derived from Human Pluripotent Stem Cells (Invited Paper).
  53. FAM43A coordinates mtDNA replication and mitochondrial biogenesis in response to mtDNA depletion.
  54. Chaperones as Potential Pharmacological Targets for Treating Protein Aggregation Illness.
  55. A primary rat neuron-astrocyte-microglia tri-culture model for studying mechanisms of neurotoxicity.
  56. Huntingtin plays an essential role in the adult hippocampus.
  57. Hypoxia-inducible factor 1 protects neurons from Sarm1-mediated neurodegeneration.
  58. Deacetylated SNAP47 recruits HOPS to facilitate autophagosome-lysosome fusion independent of STX17.
  59. The ortholog of human DNAJC9 promotes histone H3-H4 degradation and is counteracted by Asf1 in fission yeast.
  1. Neurobiol Dis. 2025 Jan 28. pii: S0969-9961(25)00031-2. [Epub ahead of print] 106815
    Sporadic ALS iPSC-derived motor neurons show axonal defects linked to altered axon guidance pathways.
    Lisha Ye, Katarina Stoklund Dittlau, Adria Sicart, Rekin''s Janky, Philip Van Damme, Ludo Van Den Bosch.
      Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disorder characterized by the selective and progressive loss of motor neurons, leading to gradual paralysis and death within 2 to 5 years after diagnosis. The exact underlying pathogenic mechanism(s) remain elusive. This is particularly the case for sporadic ALS (sALS), representing 90 % of cases, as modelling a sporadic disease is extremely difficult. We used human induced pluripotent stem cell (hiPSC)-derived motor neurons from sALS patients to investigate early disease mechanisms. The earliest phenotype that we observed were profound axonal defects including impaired axonal transport, defective axonal outgrowth and a reduced formation of neuromuscular junctions. Transcriptomic profiling revealed significant dysregulation in axon guidance pathways, with upregulation of specific axonal regeneration-inhibiting genes, such as EphA4 and DCC in sALS motor neurons. Our findings suggest that dysregulation of axon guidance pathways contributes to axonal defects and that this could play a crucial role in the pathogenesis of sALS.
    Keywords:  Amyotrophic lateral sclerosis (ALS); Axon guidance; Axonal outgrowth; Axonal transport; Human induced pluripotent stem cells (hiPSCs); Neuromuscular junctions; Sporadic ALS
    DOI:  https://doi.org/10.1016/j.nbd.2025.106815
  2. Proc Natl Acad Sci U S A. 2025 Feb 04. 122(5): e2412029122
    Endogenous LRRK2 and PINK1 function in a convergent neuroprotective ciliogenesis pathway in the brain.
    Enrico Bagnoli, Yu-En Lin, Sophie Burel, Ebsy Jaimon, Odetta Antico, Christos Themistokleous, Jonas M Nikoloff, Samuel Squires, Ilaria Morella, Jens O Watzlawik, Fabienne C Fiesel, Wolfdieter Springer, Francesca Tonelli, Pawel Lis, Simon P Brooks, Stephen B Dunnett, Riccardo Brambilla, Dario R Alessi, Suzanne R Pfeffer, Miratul M K Muqit.
      Mutations in Leucine-rich repeat kinase 2 (LRRK2) and PTEN-induced kinase 1 (PINK1) are associated with familial Parkinson's disease (PD). LRRK2 phosphorylates Rab guanosine triphosphatase (GTPases) within the Switch II domain while PINK1 directly phosphorylates Parkin and ubiquitin (Ub) and indirectly induces phosphorylation of a subset of Rab GTPases. Herein we have crossed LRRK2 [R1441C] mutant knock-in mice with PINK1 knock-out (KO) mice and report that loss of PINK1 does not impact endogenous LRRK2-mediated Rab phosphorylation nor do we see significant effect of mutant LRRK2 on PINK1-mediated Rab and Ub phosphorylation. In addition, we observe that a pool of the Rab-specific, protein phosphatase family member 1H phosphatase, is transcriptionally up-regulated and recruited to damaged mitochondria, independent of PINK1 or LRRK2 activity. Parallel signaling of LRRK2 and PINK1 pathways is supported by assessment of motor behavioral studies that show no evidence of genetic interaction in crossed mouse lines. Previously we showed loss of cilia in LRRK2 R1441C mice and herein we show that PINK1 KO mice exhibit a ciliogenesis defect in striatal cholinergic interneurons and astrocytes that interferes with Hedgehog induction of glial derived-neurotrophic factor transcription. This is not exacerbated in double-mutant LRRK2 and PINK1 mice. Overall, our analysis indicates that LRRK2 activation and/or loss of PINK1 function along parallel pathways to impair ciliogenesis, suggesting a convergent mechanism toward PD. Our data suggest that reversal of defects downstream of ciliogenesis offers a common therapeutic strategy for LRRK2 or PINK1 PD patients, whereas LRRK2 inhibitors that are currently in clinical trials are unlikely to benefit PINK1 PD patients.
    Keywords:  LRRK2; PINK1; brain; ciliogenesis; phosphorylation
    DOI:  https://doi.org/10.1073/pnas.2412029122
  3. Cell Death Discov. 2025 Jan 25. 11(1): 23
    Rapid iPSC-derived neuromuscular junction model uncovers motor neuron dominance in amyotrophic lateral sclerosis cytopathy.
    Hsiao-Chien Ting, Yun-Ting Guo, Hong-Lin Su, Yu-Shuan Chen, Shinn-Zong Lin, Horng-Jyh Harn, Chia-Yu Chang.
      The neuromuscular junction (NMJ) is essential for transmitting signals from motor neurons (MNs) to skeletal muscles (SKMs), and its dysfunction can lead to severe motor disorders. However, our understanding of the NMJ is limited by the absence of accurate human models. Although human induced pluripotent stem cell (iPSC)-derived models have advanced NMJ research, their application is constrained by challenges such as limited differentiation efficiency, lengthy generation times, and cryopreservation difficulties. To overcome these limitations, we developed a rapid human NMJ model using cryopreserved MNs and SKMs derived from iPSCs. Within 12 days of coculture, we successfully recreated NMJ-specific connectivity that closely mirrors in vivo synapse formation. Using this model, we investigated amyotrophic lateral sclerosis (ALS) and replicated ALS-specific NMJ cytopathies with SOD1 mutant and corrected isogenic iPSC lines. Quantitative analysis of 3D confocal microscopy images revealed a critical role of MNs in initiating ALS-related NMJ cytopathies, characterized by alterations in the volume, number, intensity, and distribution of acetylcholine receptors, ultimately leading to impaired muscle contractions. Our rapid and precise in vitro NMJ model offers significant potential for advancing research on NMJ physiology and pathology, as well as for developing treatments for NMJ-related diseases.
    DOI:  https://doi.org/10.1038/s41420-025-02302-5
  4. Acta Neuropathol Commun. 2025 Jan 29. 13(1): 19
    Understanding retinal tau pathology through functional 2D and 3D iPSC-derived in vitro retinal models.
    Lorenza Mautone, Federica Cordella, Alessandro Soloperto, Silvia Ghirga, Giorgia Di Gennaro, Ylenia Gigante, Silvia Di Angelantonio.
      The generation of retinal models from human induced pluripotent stem cells holds significant potential for advancing our understanding of retinal development, neurodegeneration, and the in vitro modeling of neurodegenerative disorders. The retina, as an accessible part of the central nervous system, offers a unique window into these processes, making it invaluable for both study and early diagnosis. This study investigates the impact of the Frontotemporal Dementia-linked IVS 10 + 16 MAPT mutation on retinal development and function using 2D and 3D retinal models derived from human induced pluripotent stem cells. Our findings reveal that the MAPT mutation leads to delayed retinal cell differentiation and maturation, with tau-mutant disease models exhibiting sustained higher expression of retinal progenitor cell markers and a reduced presence of post-mitotic neurons. Both 2D and 3D tau-mutant retinal models demonstrated an imbalance in tau isoforms, favoring 4R tau, along with increased tau phosphorylation, altered neurite morphology, and impaired cytoskeletal maturation. These changes are associated with impaired synaptic development, reduced neuronal connectivity, and enhanced cellular stress responses, including the increased formation of stress granules, markers of apoptosis and autophagy, and the presence of intracellular toxic tau aggregates. This study highlights the value of retinal models derived from human induced pluripotent stem cells in exploring the mechanisms underlying retinal pathology associated with tau mutations. These models offer essential insights into the development of therapeutic strategies for neurodegenerative diseases characterized by tau aggregation.
    Keywords:  Calcium imaging; Frontotemporal Dementia; Induced pluripotent stem cells; Neurodegeneration; Neurodevelopment; Organoids; Phospho-tau; Retina; Tau; Tauopathies
    DOI:  https://doi.org/10.1186/s40478-024-01920-x
  5. Cells. 2025 Jan 06. pii: 56. [Epub ahead of print]14(1):
    Small Extracellular Vesicles Promote Axon Outgrowth by Engaging the Wnt-Planar Cell Polarity Pathway.
    Samar Ahmad, Tania Christova, Melanie Pye, Masahiro Narimatsu, Siyuan Song, Jeffrey L Wrana, Liliana Attisano.
      In neurons, the acquisition of a polarized morphology is achieved upon the outgrowth of a single axon from one of several neurites. Small extracellular vesicles (sEVs), such as exosomes, from diverse sources are known to promote neurite outgrowth and thus may have therapeutic potential. However, the effect of fibroblast-derived exosomes on axon elongation in neurons of the central nervous system under growth-permissive conditions remains unclear. Here, we show that fibroblast-derived sEVs promote axon outgrowth and a polarized neuronal morphology in mouse primary embryonic cortical neurons. Mechanistically, we demonstrate that the sEV-induced increase in axon outgrowth requires endogenous Wnts and core PCP components including Prickle, Vangl, Frizzled, and Dishevelled. We demonstrate that sEVs are internalized by neurons, colocalize with Wnt7b, and induce relocalization of Vangl2 to the distal axon during axon outgrowth. In contrast, sEVs derived from neurons or astrocytes do not promote axon outgrowth, while sEVs from activated astrocytes inhibit elongation. Thus, our data reveal that fibroblast-derived sEVs promote axon elongation through the Wnt-PCP pathway in a manner that is dependent on endogenous Wnts.
    Keywords:  axon outgrowth; cortical neurons; extracellular vesicles; neurite outgrowth; planar cell polarity
    DOI:  https://doi.org/10.3390/cells14010056
  6. BMC Neurosci. 2025 Jan 28. 26(1): 8
    NPT100-18A rescues mitochondrial oxidative stress and neuronal degeneration in human iPSC-based Parkinson's model.
    Julian E Alecu, Veronika Sigutova, Razvan-Marius Brazdis, Sandra Lörentz, Marios Evangelos Bogiongko, Anara Nursaitova, Martin Regensburger, Laurent Roybon, Kerstin M Galler, Wolfgang Wrasidlo, Beate Winner, Iryna Prots.
       BACKGROUND: Parkinson's disease (PD) is a neurodegenerative disorder characterized by protein aggregates mostly consisting of misfolded alpha-synuclein (αSyn). Progressive degeneration of midbrain dopaminergic neurons (mDANs) and nigrostriatal projections results in severe motor symptoms. While the preferential loss of mDANs has not been fully understood yet, the cell type-specific vulnerability has been linked to a unique intracellular milieu, influenced by dopamine metabolism, high demand for mitochondrial activity, and increased level of oxidative stress (OS). These factors have been shown to adversely impact αSyn aggregation. Reciprocally, αSyn aggregates, in particular oligomers, can impair mitochondrial functions and exacerbate OS. Recent drug-discovery studies have identified a series of small molecules, including NPT100-18A, which reduce αSyn oligomerization by preventing misfolding and dimerization. NPT100-18A and structurally similar compounds (such as NPT200-11/UCB0599, currently being assessed in clinical studies) point towards a promising new approach for disease-modification.
    METHODS: Induced pluripotent stem cell (iPSC)-derived mDANs from PD patients with a monoallelic SNCA locus duplication and unaffected controls were treated with NPT100-18A. αSyn aggregation was evaluated biochemically and reactive oxygen species (ROS) levels were assessed in living mDANs using fluorescent dyes. Adenosine triphosphate (ATP) levels were measured using a luminescence-based assay, and neuronal cell death was evaluated by immunocytochemistry.
    RESULTS: Compared to controls, patient-derived mDANs exhibited higher cytoplasmic and mitochondrial ROS probe levels, reduced ATP-related signals, and increased activation of caspase-3, reflecting early neuronal cell death. NPT100-18A-treatment rescued cleaved caspase-3 levels to control levels and, importantly, attenuated mitochondrial oxidative stress probe levels in a compartment-specific manner and, at higher concentrations, increased ATP signals.
    CONCLUSIONS: Our findings demonstrate that NPT100-18A limits neuronal degeneration in a human in vitro model of PD. In addition, we provide first mechanistic insights into how a compartment-specific antioxidant effect in mitochondria might contribute to the neuroprotective effects of NPT100-18A.
    Keywords:  Aggregation; Alpha-synuclein; Dopaminergic neurons; Mitochondria; NPT100-18A; Oxidative stress; Parkinson’s disease; ROS; iPSC
    DOI:  https://doi.org/10.1186/s12868-025-00926-y
  7. PLoS Genet. 2025 Jan;21(1): e1011547
    Tail Anchored protein insertion mediated by CAML and TRC40 links to neuromuscular function in mice.
    Ying Zhang, Lihong He, Justin Gundelach, Anjie Ge, Helena Edlund, Stefan Norlin, Richard J Bram.
      Motor neuron diseases, such as amyotrophic lateral sclerosis (ALS) and progressive bulbar palsy, involve loss of muscle control resulting from death of motor neurons. Although the exact pathogenesis of these syndromes remains elusive, many are caused by genetically inherited mutations. Thus, it is valuable to identify additional genes that can impact motor neuron survival and function. In this report, we describe mice that express globally reduced levels of calcium-modulating cyclophilin ligand (CAML) protein. CAML is an essential component in the transmembrane domain recognition complex (TRC) pathway, responsible for inserting C-terminal tail anchored (TA) proteins into the endoplasmic reticulum membrane. The primary phenotype observed in these mice was rapid development of hind limb weakness and paralysis. Spinal cord sections revealed a loss of motor neuron cell bodies. Targeting CAML loss specifically to neurons using SLICK-H-Cre or synapsin-Cre transgenic mice yielded similar phenotypes, indicating that CAML plays a cell autonomous role in this process. We found that intracellular trafficking was perturbed in cells depleted of CAML, with aberrant release of procathepsin D and defective retention of CD222 within the trans-Golgi network, as well as reduced levels and mislocalization of syntaxin 5 (Stx5). Dysfunctional lysosomes and abnormal protein glycosylation were also revealed in CAML deficient cells, further indicating a defect in Golgi trafficking. In addition, we observed an identical phenotype in mice lacking ASNA1 in neurons, suggesting that CAML's role in sustaining muscle function is related to its involvement in the TRC pathway. Together, these findings implicate motor neuron survival as a key role for the TA protein insertion machinery in mice, which may shed light on the pathogenesis of neuromuscular disease in humans.
    DOI:  https://doi.org/10.1371/journal.pgen.1011547
  8. Mol Ther Nucleic Acids. 2025 Mar 11. 36(1): 102441
    Modeling and correction of protein conformational disease in iPSC-derived neurons through personalized base editing.
    Colin T Konishi, Nancy Mulaiese, Tanvi Butola, Qinkun Zhang, Dana Kagan, Qiaoyan Yang, Mariel Pressler, Brooke G Dirvin, Orrin Devinsky, Jayeeta Basu, Chengzu Long.
      Altered protein conformation can cause incurable neurodegenerative disorders. Mutations in SERPINI1, the gene encoding neuroserpin, can alter protein conformation resulting in cytotoxic aggregation leading to neuronal death. Familial encephalopathy with neuroserpin inclusion bodies (FENIB) is a rare autosomal dominant progressive myoclonic epilepsy that progresses to dementia and premature death. We developed HEK293T and induced pluripotent stem cell (iPSC) models of FENIB, harboring a patient-specific pathogenic SERPINI1 variant or stably overexpressing mutant neuroserpin fused to GFP (MUT NS-GFP). Here, we utilized a personalized adenine base editor (ABE)-mediated approach to correct the pathogenic variant efficiently and precisely to restore neuronal dendritic morphology. ABE-treated MUT NS-GFP cells demonstrated reduced inclusion size and number. Using an inducible MUT NS-GFP neuron system, we identified early prevention of toxic protein expression allowed aggregate clearance, while late prevention halted further aggregation. To address several challenges for clinical applications of gene correction, we developed a neuron-specific engineered virus-like particle to optimize neuronal ABE delivery, resulting in higher correction efficiency. Our findings provide a targeted strategy that may treat FENIB and potentially other neurodegenerative diseases due to altered protein conformation such as Alzheimer's and Huntington's diseases.
    Keywords:  CRISPR-Cas9; MT: RNA/DNA Editing; adenine base editing; engineered virus-like particles; iPSC-derived neurons; protein aggregation; protein conformational disease
    DOI:  https://doi.org/10.1016/j.omtn.2024.102441
  9. Brain Behav. 2025 Jan;15(1): e70256
    Prognostic Value of Cerebrospinal Fluid and Serum Neurofilament Light Chain in Amyotrophic Lateral Sclerosis: A Correlation Study.
    Siqi Dong, Xiaoni Liu, Yanni Zhou, Jiatong Li, Zihan Qi, Zihan Wang, Wenbo Yang, Xiangjun Chen.
       BACKGROUND: The diagnostic and prognostic values of serum neurofilament light chain (sNfL), in comparison to cerebrospinal fluid (CSF) neurofilament light chain (cNfL), and other clinical parameters in amyotrophic lateral sclerosis (ALS) at the time of diagnosis remain elusive.
    METHODS: We examine paired serum and CSF samples from 80 ALS patients and 21 control subjects, all obtained at the time of diagnosis. Additional serum samples were collected from 51 other ALS patients. NfL concentrations were quantified using the single molecule array (Simoa) technique.
    RESULTS: Our findings demonstrate a robust correlation between NfL levels in matched CSF and serum samples. Notably, both sNfL (p < 0.0001) and cNfL (p < 0.0001) exhibited significantly elevated levels in ALS patients compared to controls. Furthermore, baseline sNfL concentrations, as well as cNfL levels, emerged as predictive indicators of subsequent disease progression rate (sNfL: p < 0.0001, cNfL: p = 0.0005) and overall survival (sNfL: p = 0.0073, cNfL: p = 0.0044). Employing a Cox regression model, we identified baseline sNfL level (HR = 1.01, p = 0.013), and diagnostic delay (HR = 0.94, p = 0.003) as independent prognostic factors for mortality. Furthermore, we constructed a nomogram model that incorporates both sNfL and pertinent clinical variables, which substantially enhances the accuracy of predicting disease outcomes (Concordance Index, 0.808).
    CONCLUSION: Our study underscores the robust correlation between sNfL and cNfL in ALS patients and establishes baseline sNfL as a potent and independent prognostic marker for mortality.
    Keywords:  amyotrophic lateral sclerosis; biomarker; diagnosis; neurofilament light chain; prognosis
    DOI:  https://doi.org/10.1002/brb3.70256
  10. Life Sci Alliance. 2025 Apr;pii: e202402907. [Epub ahead of print]8(4):
    A role for mitochondria-ER crosstalk in amyotrophic lateral sclerosis 8 pathogenesis.
    Cathal Wilson, Laura Giaquinto, Michele Santoro, Giuseppe Di Tullio, Valentina Morra, Wanda Kukulski, Rossella Venditti, Francesca Navone, Nica Borgese, Maria Antonietta De Matteis.
      Protein aggregates in motoneurons, a pathological hallmark of amyotrophic lateral sclerosis, have been suggested to play a key pathogenetic role. ALS8, characterized by ER-associated inclusions, is caused by a heterozygous mutation in VAPB, which acts at multiple membrane contact sites between the ER and almost all other organelles. The link between protein aggregation and cellular dysfunction is unclear. A yeast model, expressing human mutant and WT-VAPB under the control of the orthologous yeast promoter in haploid and diploid cells, was developed to mimic the disease situation. Inclusion formation was found to be a developmentally regulated process linked to mitochondrial damage that could be attenuated by reducing ER-mitochondrial contacts. The co-expression of the WT protein retarded P56S-VAPB inclusion formation. Importantly, we validated these results in mammalian motoneuron cells. Our findings indicate that (age-related) damage to mitochondria influences the propensity of the mutant VAPB to form aggregates via ER-mitochondrial contacts, initiating a series of events leading to disease progression.
    DOI:  https://doi.org/10.26508/lsa.202402907
  11. Lancet Neurol. 2025 Feb;pii: S1474-4422(24)00517-9. [Epub ahead of print]24(2): 166-178
    Amyotrophic lateral sclerosis caused by FUS mutations: advances with broad implications.
    Thomas G Moens, Sandrine Da Cruz, Manuela Neumann, Tatyana A Shelkovnikova, Neil A Shneider, Ludo Van Den Bosch.
      Autosomal dominant mutations in the gene encoding the DNA and RNA binding protein FUS are a cause of amyotrophic lateral sclerosis (ALS), and about 0·3-0·9% of patients with ALS are FUS mutation carriers. FUS-mutation-associated ALS (FUS-ALS) is characterised by early onset and rapid progression, compared with other forms of ALS. However, different pathogenic mutations in FUS can result in markedly different age at symptom onset and rate of disease progression. Most FUS mutations disrupt its nuclear localisation, leading to its cytoplasmic accumulation in the CNS. FUS also forms inclusions in around 5% of patients with the related neurodegenerative condition frontotemporal dementia. However, there are key differences between the two diseases at the genetic and neuropathological level, which suggest distinct pathogenic processes. Experimental models have uncovered potential pathogenic mechanisms in FUS-ALS and informed therapeutic strategies that are currently in development, including the silencing of FUS expression using an intrathecally administered antisense oligonucleotide.
    DOI:  https://doi.org/10.1016/S1474-4422(24)00517-9
  12. Biochem Biophys Res Commun. 2025 Jan 25. pii: S0006-291X(25)00109-3. [Epub ahead of print]750 151395
    Intramuscular inhibition of glycogen phosphorylase improves motor function in spinal cord injury.
    Ximeng Yang, Maho Kondo, Chihiro Tohda.
      Motor dysfunction in various diseases and aging is often accompanied by skeletal muscle atrophy and reduced axonal projections from motor neurons to the skeletal muscles. While several studies have investigated the correlations and molecular mechanisms between muscle atrophy and motor neuron denervation to explain the pathology of motor diseases, it remains unclear whether skeletal muscle atrophy directly causes axonal denervation of motor neurons. Here, we used a casts-attached mouse model which represents muscle atrophy and motor dysfunction in the hindlimbs to explore how skeletal muscle atrophy affects motor neuronal axon projections. Retrograde neuronal tracing from the skeletal muscles to motor neurons revealed that axonal projections from motor neurons were reduced to the atrophied skeletal muscles compared to the healthy muscles. In addition, we identified glycogen phosphorylase (GP) as an upregulated protein in the plasma membrane of atrophied gastrocnemius muscles. The expression level of GP was also increased on the membrane of primary cultured myotubes treated with dexamethasone to induce muscle atrophy in vitro. Importantly, intramuscular injection of a GP inhibitor into the hindlimbs improved motor function in a mouse model of spinal cord injury. Furthermore, axonal projection from spinal cord neurons to dexamethasone-treated atrophied myotubes was reduced compared to healthy myotubes, whereas GP inhibitor treatment to atrophied myotubes promoted axonal growth of the spinal cord neurons overlayed on the myotubes. This study demonstrated that skeletal muscle atrophy induces attenuation of motor neuronal innervation and inhibition of GP in atrophied skeletal muscles may be a novel therapeutic approach for spinal cord injury by enhancing axonal projections from motor neurons to the skeletal muscles.
    Keywords:  Axonal projection; Glycogen phosphorylase; Motor neurons; Muscle atrophy; Spinal cord injury
    DOI:  https://doi.org/10.1016/j.bbrc.2025.151395
  13. FEBS J. 2025 Jan 31.
    A cellular model of TDP-43 induces phosphorylated TDP-43 aggregation with distinct changes in solubility and autophagy dysregulation.
    Matthew B Dopler, Muhammad I Abeer, Sanaz Arezoumandan, Keyshawn Cox, Tyler L Petersen, Esther H Daniel, Carlton L Cannon, Angelica Bautista, Kennedy D Blancher, Alysia M Bland, Kylie J Bond, Dominque A Davis, Jessica M Francois, Eliana J McCray, Justin M Morgan, Jessica L Pulliam, Zymir A Robinson, Mykia J Taylor, James A Dowell, Nigel J Cairns, Michael A Gitcho.
      Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease that affects neurons in the brain and spinal cord, causing loss of muscle control, and eventually leads to death. Phosphorylated transactive response DNA binding protein-43 (TDP-43) is the major pathological protein in both sporadic and familial ALS, forming cytoplasmic aggregates in over 95% of cases. Of the 10-15% of ALS cases that are familial, mutations in TDP-43 represent about 5% of those with a family history. We have developed an in vitro overexpression model by introducing three familial ALS mutations (A315T, M337V, and S379P) in the TDP-43 (TARDBP) gene which we define as 3X-TDP-43. This overexpression model TDP-43 shows deficits in autophagy flux and colocalization of TDP-43 with stress granules. We also observe a progressive shift of TDP-43 to the cytoplasm in this model. This overexpression model shows a reduction in solubility of phosphorylated TDP-43 from RIPA to urea soluble. Four glycolytic enzymes, phosphoglycerate kinase one (PGK1), aldolase A (ALDOA), enolase 1 (ENO1), and pyruvate dehydrogenase kinase 1 (PDK1) show significant time-dependent decreases in 3X-TDP-43 expressing cells. Shotgun proteomic analysis shows global changes in the importin subunit alpha-1 (KPNA2), heat shock 70 kDa protein 1A (HSPA1A), and protein disulfide-isomerase A3 (PDIA3) expression levels and coimmunoprecipitation reveals that these proteins complex with TDP-43. Overall, these results suggest that the 3X-TDP-43 model may provide new insights into pathophysiology and an avenue for drug screening in vitro for those suffering from ALS and related TDP-43 proteinopathies.
    Keywords:  ALS; TDP‐43; aggregation; autophagy; glycolysis; stress granules
    DOI:  https://doi.org/10.1111/febs.17413
  14. Cell Transplant. 2025 Jan-Dec;34:34 9636897241303271
    Advances in the Development and Application of Human Organoids: Techniques, Applications, and Future Perspectives.
    Zhangcheng Zhu, Yiwen Cheng, Xia Liu, Wenwen Ding, Jiaming Liu, Zongxin Ling, Lingbin Wu.
      Organoids are three-dimensional (3D) cell cultures derived from human pluripotent stem cells or adult stem cells that recapitulate the cellular heterogeneity, structure, and function of human organs. These microstructures are invaluable for biomedical research due to their ability to closely mimic the complexity of native tissues while retaining human genetic material. This fidelity to native organ systems positions organoids as a powerful tool for advancing our understanding of human biology and for enhancing preclinical drug testing. Recent advancements have led to the successful development of a variety of organoid types, reflecting a broad range of human organs and tissues. This progress has expanded their application across several domains, including regenerative medicine, where organoids offer potential for tissue replacement and repair; disease modeling, which allows for the study of disease mechanisms and progression in a controlled environment; drug discovery and evaluation, where organoids provide a more accurate platform for testing drug efficacy and safety; and microecological research, where they contribute to understanding the interactions between microbes and host tissues. This review provides a comprehensive overview of the historical development of organoid technology, highlights the key achievements and ongoing challenges in the field, and discusses the current and emerging applications of organoids in both laboratory research and clinical practice.
    Keywords:  disease modeling; induced pluripotent stem cells; microbiota; organoids; regenerative medicine
    DOI:  https://doi.org/10.1177/09636897241303271
  15. Commun Biol. 2025 Jan 28. 8(1): 136
    TDP-43 nuclear retention is antagonized by hypo-phosphorylation of its C-terminus in the cytoplasm.
    Célia Rabhi, Nicolas Babault, Céline Martin, Bénédicte Desforges, Alexandre Maucuer, Vandana Joshi, Serhii Pankivskyi, Yitian Feng, Guillaume Bollot, Revital Rattenbach, David Pastré, Ahmed Bouhss.
      Protein aggregation is a hallmark of many neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), in which TDP-43, a nuclear RNA-binding protein, forms cytoplasmic inclusions. Here, we have developed a robust and automated method to assess protein self-assembly in the cytoplasm using microtubules as nanoplatforms. Importantly, we have analyzed specifically the self-assembly of full-length TDP-43 and its mRNA binding that are regulated by the phosphorylation of its self-adhesive C-terminus, which is the recipient of many pathological mutations. We show that C-terminus phosphorylation prevents the recruitment of TDP-43 in mRNA-rich stress granules only under acute stress conditions because of a low affinity for mRNA but not under mild stress conditions. In addition, the self-assembly of the C-terminus is negatively regulated by phosphorylation in the cytoplasm which in turn promotes TDP-43 nuclear import. We anticipate that reducing TDP-43 C-terminus self-assembly in the cytoplasm may be an interesting strategy to reverse TDP-43 nuclear depletion in neurodegenerative diseases.
    DOI:  https://doi.org/10.1038/s42003-025-07456-7
  16. Adv Drug Deliv Rev. 2025 Jan 27. pii: S0169-409X(25)00010-9. [Epub ahead of print]218 115525
    Clinical perspective on pluripotent stem cells derived cell therapies for the treatment of neurodegenerative diseases.
    Michal Izrael, Judith Chebath, Kfir Molakandov, Michel Revel.
      Self-renewal capacity and potential to differentiate into almost any cell type of the human body makes pluripotent stem cells a valuable starting material for manufacturing of clinical grade cell therapies. Neurodegenerative diseases are characterized by gradual loss of structure or function of neurons, often leading to neuronal death. This results in gradual decline of cognitive, motor, and physiological functions due to the degeneration of the central nervous systems. Over the past two decades, comprehensive preclinical efficacy (proof-of-concept) and safety studies have led to the initiation of First-in-Human phase I-II clinical trials for a range of neurodegenerative diseases. In this review, we explore the fundamentals and challenges of neural-cell therapies derived from pluripotent stem cells for treating neurodegenerative diseases. Additionally, we highlight key preclinical investigations that paved the way for regulatory approvals of these trials. Furthermore, we provide an overview on progress and status of clinical trials done so far in treating neurodegenerative diseases such as spinal cord injury (SCI), Parkinson's disease (PD), and amyotrophic lateral sclerosis (ALS), as well as advances in retina diseases such as Stargardt disease (a.k.a fundus flavimaculatus), retinitis pigmentosa (RP) and age-related macular degeneration (AMD). These trials will pave the way for the development of new cell-based therapies targeting additional neurological conditions, including Alzheimer's disease and epilepsy.
    Keywords:  Astrocytes; Dopaminergic neurons; Embryonic stem cells; Induced pluripotent stem cells; Pluripotent stem cells; Retina pigmented epithelial cell
    DOI:  https://doi.org/10.1016/j.addr.2025.115525
  17. J Clin Med. 2025 Jan 16. pii: 545. [Epub ahead of print]14(2):
    Resting-State EEG Oscillations in Amyotrophic Lateral Sclerosis (ALS): Toward Mechanistic Insights and Clinical Markers.
    James Chmiel, Marta Stępień-Słodkowska.
      Introduction: Amyotrophic lateral sclerosis (ALS) is a complex, progressive neurodegenerative disorder characterized by the degeneration of motor neurons in the brain, brainstem, and spinal cord. Several neuroimaging techniques can help reveal the pathophysiology of ALS. One of these is the electroencephalogram (EEG), a noninvasive and relatively inexpensive tool for examining electrical activity of the brain with excellent temporal precision. Methods: This mechanistic review examines the pattern of resting-state EEG activity. With a focus on publications published between January 1995 and October 2024, we carried out a comprehensive search in October 2024 across a number of databases, including PubMed/Medline, Research Gate, Google Scholar, and Cochrane. Results: The literature search yielded 17 studies included in this review. The studies varied significantly in their methodology and patient characteristics. Despite this, a common biomarker typical of ALS was found-reduced alpha power. Regarding other oscillations, the findings are less consistent and sometimes contradictory. As this is a mechanistic review, three possible explanations for this biomarker are provided. The main and most important one is increased cortical excitability. In addition, due to the limitations of the studies, recommendations for future research on this topic are outlined to enable a further and better understanding of EEG patterns in ALS. Conclusions: Most studies included in this review showed alpha power deficits in ALS patients, reflecting pathological hyperexcitability of the cerebral cortex. Future studies should address the methodological limitations identified in this review, including small sample sizes, inconsistent frequency-band definitions, and insufficient functional outcome measures, to solidify and extend current findings.
    Keywords:  ALS; EEG; QEEG; amyotrophic lateral sclerosis; electroencephalogram; electroencephalography; electrophysiology; neural correlates; neuroimaging; neurophysiology; oscillations
    DOI:  https://doi.org/10.3390/jcm14020545
  18. Int J Mol Sci. 2024 Dec 30. pii: 230. [Epub ahead of print]26(1):
    Systematic Evaluation of Extracellular Coating Matrix on the Differentiation of Human-Induced Pluripotent Stem Cells to Cortical Neurons.
    Siyao Li, Yan Liu, Xianyang Luo, Wei Hong.
      Induced pluripotent stem cell (iPSC)-derived neurons (iNs) have been widely used as models of neurodevelopment and neurodegenerative diseases. Coating cell culture vessels with extracellular matrixes (ECMs) gives structural support and facilitates cell communication and differentiation, ultimately enhances neuronal functions. However, the relevance of different ECMs to the natural environment and their impact on neuronal differentiation have not been fully characterized. In this study, we report the use of four commonly used extracellular matrixes, poly-D-lysine (PDL), poly-L-ornithine (PLO), Laminin and Matrigel, which we applied to compare the single-coating and double-coating conditions on iNs differentiation and maturation. Using the IncuCyte live-cell imaging system, we found that iNs cultured on single Matrigel- and Laminin-coated vessels have significantly higher density of neurite outgrowth and branch points than PLO or PDL but produce abnormal highly straight neurite outgrowth and larger cell body clumps. All the four double-coating conditions significantly reduced the clumping of neurons, in which the combination of PDL+Matrigel also enhanced neuronal purity. Double coating with PDL+Matrigel also tended to improve dendritic and axonal development and the distribution of pre and postsynaptic markers. These results demonstrate that the extracellular matrix contributes to the differentiation of cultured neurons and that double coating with PDL+Matrigel gives the best outcomes. Our study indicates that neuronal differentiation and maturation can be manipulated, to a certain extent, by adjusting the ECM recipe, and provides important technical guidance for the use of the ECM in neurological studies.
    Keywords:  induced pluripotent stem cells (iPSCs); live-cell imaging; morphology; neurite outgrowth; neuronal differentiation; synaptic markers
    DOI:  https://doi.org/10.3390/ijms26010230
  19. Proc Natl Acad Sci U S A. 2025 Feb 04. 122(5): e2404017121
    Cryo-ET suggests tubulin chaperones form a subset of microtubule lumenal particles with a role in maintaining neuronal microtubules.
    Saikat Chakraborty, Antonio Martinez-Sanchez, Florian Beck, Mauricio Toro-Nahuelpan, In-Young Hwang, Kyung-Min Noh, Wolfgang Baumeister, Julia Mahamid.
      The functional architecture of the long-lived neuronal microtubule (MT) cytoskeleton is maintained by various MT-associated proteins (MAPs), most of which are known to bind to the MT outer surface. However, electron microscopy (EM) has long ago revealed the presence of particles inside the lumens of neuronal MTs, of yet unknown identity and function. Here, we use cryogenic electron tomography (cryo-ET) to analyze the three-dimensional (3D) organization and structures of MT lumenal particles in primary hippocampal neurons, human induced pluripotent stem cell-derived neurons, and pluripotent and differentiated P19 cells. We obtain in situ density maps of several lumenal particles from the respective cells and detect common structural features underscoring their potential overarching functions. Mass spectrometry-based proteomics combined with structural modeling suggest that a subset of lumenal particles could be tubulin-binding cofactors (TBCs) bound to tubulin monomers. A different subset of smaller particles, which remains unidentified, exhibits densities that bridge across the MT protofilaments. We show that increased lumenal particle concentration within MTs is concomitant with neuronal differentiation and correlates with higher MT curvatures. Enrichment of lumenal particles around MT lattice defects and at freshly polymerized MT open-ends suggests a MT protective role. Together with the identified structural resemblance of a subset of particles to TBCs, these results hint at a role in local tubulin proteostasis for the maintenance of long-lived neuronal MTs.
    Keywords:  hiPSC-derived neurons; in situ cryoelectron tomography; microtubule lattice damage; primary neurons; subtomogram averaging
    DOI:  https://doi.org/10.1073/pnas.2404017121
  20. Alzheimers Dement. 2025 Jan;21(1): e14560
    APP antisense oligonucleotides are effective in rescuing mitochondrial phenotypes in human iPSC-derived trisomy 21 astrocytes.
    Srishruthi Thirumalai, Frederick J Livesey, Rickie Patani, Christy Hung.
       INTRODUCTION: Antisense oligonucleotides (ASOs) have shown promise in reducing amyloid precursor protein (APP) levels in neurons, but their effects in astrocytes, key contributors to neurodegenerative diseases, remain unclear. This study evaluates the efficacy of APP ASOs in astrocytes derived from an individual with Down syndrome (DS), a population at high risk for Alzheimer's disease (AD).
    METHODS: Human induced pluripotent stem cells (hiPSCs) from a healthy individual and an individual with DS were differentiated into astrocytes. Astrocytes were treated with APP ASOs for 10 days, and APP levels were quantified. Mitochondrial morphology and superoxide production in DS astrocytes were analyzed using super-resolution and confocal microscopy.
    RESULTS: APP ASOs significantly reduced APP levels in astrocytes from both control and DS individuals. In DS astrocytes, treatment restored mitochondrial health, increasing mitochondrial number and size while reducing superoxide production.
    DISCUSSION: APP ASOs effectively reduce APP levels and improve mitochondrial health in astrocytes, suggesting their potential as a therapeutic approach for DS and DS-related AD. Further in vivo studies are required to confirm these findings.
    HIGHLIGHTS: APP ASOs reduce APP levels in human iPSC-derived astrocytes. APP ASO treatment rescues mitochondrial phenotypes in trisomy 21 astrocytes. This study supports ASOs as a potential therapy for Down syndrome-related Alzheimer's disease.
    Keywords:  APP; Alzheimer's disease; Down syndrome; antisense oligonucleotides; astrocytes; mitochondrial function
    DOI:  https://doi.org/10.1002/alz.14560
  21. Nat Commun. 2025 Jan 28. 16(1): 1109
    Activation of lysophagy by a TBK1-SCFFBXO3-TMEM192-TAX1BP1 axis in response to lysosomal damage.
    Na Yeon Park, Doo Sin Jo, Jae-Yoon Yang, Ji-Eun Bae, Joon Bum Kim, Yong Hwan Kim, Seong Hyun Kim, Pansoo Kim, Dong-Seok Lee, Tamotsu Yoshimori, Eun-Kyeong Jo, Eunbyul Yeom, Dong-Hyung Cho.
      Lysophagy eliminates damaged lysosomes and is crucial to cellular homeostasis; however, its underlying mechanisms are not entirely understood. We screen a ubiquitination-related compound library and determine that the substrate recognition component of the SCF-type E3 ubiquitin ligase complex, SCFFBXO3(FBXO3), which is a critical lysophagy regulator. Inhibition of FBXO3 reduces lysophagy and lysophagic flux in response to L-leucyl-L-leucine methyl ester (LLOMe). Furthermore, FBXO3 interacts with TMEM192, leading to its ubiquitination in LLOMe-treated cells. We also identify TAX1BP1 as a critical autophagic adaptor that recognizes ubiquitinated TMEM192 during lysophagy and find that TBK1 activation is crucial for lysophagy, as it phosphorylates FBXO3 in response to lysosomal damage. Knockout of FBXO3 significantly impairs lysophagy, and its reconstitution with a loss-of-function mutant (V221I) further confirms its essential role in lysophagy regulation. Collectively, our findings highlight the significance of the TBK1-FBXO3-TMEM192-TAX1BP1 axis in lysophagy and emphasize the critical role of FBXO3 in lysosomal integrity.
    DOI:  https://doi.org/10.1038/s41467-025-56294-y
  22. Stem Cell Rev Rep. 2025 Jan 31.
    Optimization of Transcription Factor-Driven Neuronal Differentiation from Human Induced Pluripotent Stem Cells for Disease Modelling and Drug Screening.
    Martina Servetti, Martino Caramia, Giulia Parodi, Fabrizio Loiacono, Ennio Nano, Giorgia Biddau, Lorenzo Ferrando, Lisastella Morinelli, Pierluigi Valente, Sergio Martinoia, Andrea Escelsior, Gianluca Serafini, Serena Tamburro, Simona Baldassari, Anna Fassio, Fabio Benfenati, Anna Corradi, Bruno Sterlini.
      Progress of human brain in vitro models stands as a keystone in neurological and psychiatric research, addressing the limitations posed by species-specific differences in animal models. The generation of human neurons from induced pluripotent stem cells (iPSCs) using transcription factor reprogramming protocols has been shown to reduce heterogeneity and improve consistency across different stem cell lines. Despite notable advancements, the current protocols still exhibit several shortcomings. This study focuses on standardizing and optimizing the procedure for iPSC-derived glutamatergic neurons generation through the inducible overexpression of Neurogenin-2. Noteworthy refinements include stringent scrutiny of genomic rearrangements post-fibroblast reprogramming, selection of a homogeneously integrated NGN2-cassettes population, and the incorporation of an intermediate step during neuronal differentiation to store neuronal progenitors. The neural culture showed a high degree of neuronal maturation and consistency, as shown by single-cell and network electrophysiological recordings. These advancements aim to provide more reliable tools for disease modelling and drug screening in neurological disorders.
    Keywords:  Electrophysiological recordings; FACS sorting; NGN2-mediated neuronal differentiation; Transcription factor–driven differentiation; iGluNeurons; iPSCs
    DOI:  https://doi.org/10.1007/s12015-025-10845-4
  23. Lancet Neurol. 2025 Feb;pii: S1474-4422(24)00523-4. [Epub ahead of print]24(2): 100-101
    Transforming amyotrophic lateral sclerosis into a liveable disease.
    Eva L Feldman, Rita Sattler, Matthew C Kiernan, Stephen A Goutman, Adriano Chiò, Ammar Al-Chalabi.
      
    DOI:  https://doi.org/10.1016/S1474-4422(24)00523-4
  24. Cell Death Dis. 2025 Jan 25. 16(1): 40
    Ferroptosis triggers mitochondrial fragmentation via Drp1 activation.
    Lohans Pedrera, Laura Prieto Clemente, Alina Dahlhaus, Sara Lotfipour Nasudivar, Sofya Tishina, Daniel Olmo González, Jenny Stroh, Fatma Isil Yapici, Randhwaj Pratap Singh, Nils Grotehans, Thomas Langer, Ana J García-Sáez, Silvia von Karstedt.
      Constitutive mitochondrial dynamics ensure quality control and metabolic fitness of cells, and their dysregulation has been implicated in various human diseases. The large GTPase Dynamin-related protein 1 (Drp1) is intimately involved in mediating constitutive mitochondrial fission and has been implicated in mitochondrial cell death pathways. During ferroptosis, a recently identified type of regulated necrosis driven by excessive lipid peroxidation, mitochondrial fragmentation has been observed. Yet, how this is regulated and whether it is involved in ferroptotic cell death has remained unexplored. Here, we provide evidence that Drp1 is activated upon experimental induction of ferroptosis and promotes cell death execution and mitochondrial fragmentation. Using time-lapse microscopy, we found that ferroptosis induced mitochondrial fragmentation and loss of mitochondrial membrane potential, but not mitochondrial outer membrane permeabilization. Importantly, Drp1 accelerated ferroptotic cell death kinetics. Notably, this function was mediated by the regulation of mitochondrial dynamics, as overexpression of Mitofusin 2 phenocopied the effect of Drp1 deficiency in delaying ferroptosis cell death kinetics. Mechanistically, we found that Drp1 is phosphorylated and activated after induction of ferroptosis and that it translocates to mitochondria. Further activation at mitochondria through the phosphatase PGAM5 promoted ferroptotic cell death. Remarkably, Drp1 depletion delayed mitochondrial and plasma membrane lipid peroxidation. These data provide evidence for a functional role of Drp1 activation and mitochondrial fragmentation in the acceleration of ferroptotic cell death, with important implications for targeting mitochondrial dynamics in diseases associated with ferroptosis.
    DOI:  https://doi.org/10.1038/s41419-024-07312-2
  25. Sci Signal. 2025 Jan 21. 18(870): eadn9868
    Biotin mitigates the development of manganese-induced, Parkinson's disease-related neurotoxicity in Drosophila and human neurons.
    Yunjia Lai, Pablo Reina-Gonzalez, Gali Maor, Gary W Miller, Souvarish Sarkar.
      Chronic exposure to manganese (Mn) induces manganism and has been widely implicated as a contributing environmental factor to Parkinson's disease (PD), featuring notable overlaps between the two in motor symptoms and clinical hallmarks. Here, we developed an adult Drosophila model of Mn toxicity that recapitulated key parkinsonian features, spanning behavioral deficits, neuronal loss, and dysfunctions in lysosomes and mitochondria. Metabolomics analysis of the brain and body tissues of these flies at an early stage of toxicity identified systemic changes in the metabolism of biotin (also known as vitamin B7) in Mn-treated groups. Biotinidase-deficient flies showed exacerbated Mn-induced neurotoxicity, parkinsonism, and mitochondrial dysfunction. Supplementing the diet of wild-type flies with biotin ameliorated the pathological phenotypes of concurrent exposure to Mn. Biotin supplementation also ameliorated the pathological phenotypes of three standard fly models of PD. Furthermore, supplementing the culture media of human induced stem cells (iPSCs) differentiated midbrain dopaminergic neurons with biotin protected against Mn-induced mitochondrial dysregulation, cytotoxicity, and neuronal loss. Last, analysis of the expression of genes encoding biotin-related proteins in patients with PD revealed increased amounts of biotin transporters in the substantia nigra compared with healthy controls, suggesting a potential role of altered biotin metabolism in PD. Together, our findings identified changes in biotin metabolism as underlying Mn neurotoxicity and parkinsonian pathology in flies, for which dietary biotin supplementation was preventative.
    DOI:  https://doi.org/10.1126/scisignal.adn9868
  26. J Cell Sci. 2025 Jan 29. pii: jcs.263685. [Epub ahead of print]
    Mitochondria-plasma membrane contact sites regulate the ER-mitochondria encounter structure.
    Jason C Casler, Clare S Harper, Laura L Lackner.
      Cells form multiple, molecularly distinct membrane contact sites (MCSs) between organelles. Despite knowing the molecular identity of several of these complexes, little is known about how MCSs are coordinately regulated in space and time to promote organelle function. Here, we examined two well-characterized mitochondria-ER MCSs - the ER-Mitochondria encounter structure (ERMES) and the mitochondria-ER-cortex anchor (MECA). We report that loss of MECA results in a substantial reduction in the number of ERMES contacts. Rather than reducing ERMES protein levels, loss of MECA results in an increase in the size of ERMES contacts. Using live cell microscopy, we demonstrate that ERMES contacts display several dynamic behaviors, such as de novo formation, fusion, and fission, that are altered in the absence of MECA or by changes in growth conditions. Unexpectedly, we find that the mitochondria-PM tethering, not the mitochondria-ER tethering, function of MECA regulates ERMES contacts. Remarkably, synthetic tethering of mitochondria to the PM in the absence of MECA is sufficient to rescue the distribution of ERMES foci. Overall, our work reveals how one MCS can influence the regulation and function of another.
    Keywords:  Membrane contact sites; Mitochondria; Mitochondrial positioning
    DOI:  https://doi.org/10.1242/jcs.263685
  27. J Cell Physiol. 2025 Jan;240(1): e31523
    Glucose Transporter 1 Deficiency Impairs Glucose Metabolism and Barrier Induction in Human Induced Pluripotent Stem Cell-Derived Astrocytes.
    Iqra Pervaiz, Yash Mehta, Abraham Jacob Al-Ahmad.
      Glucose is a major source of energy for the brain. At the blood-brain barrier (BBB), glucose uptake is facilitated by glucose transporter 1 (GLUT1). GLUT1 Deficiency Syndrome (GLUT1DS), a haploinsufficiency affecting SLC2A1, reduces glucose brain uptake. A lot of effort has been made to characterize GLUT1DS at the BBB, but the impact on astrocytes remains unclear. In this study, we investigated the impact of GLUT1DS on astrocyte differentiation and function in vitro, using human induced pluripotent stem cells GLUT1DS (GLUT1DS-iPSCs) differentiated into astrocyte-like cells (iAstros). GLUT1 expression is decreased during the differentiation of iPSCs into astrocytes, with neural progenitor cells showing the lowest expression. The presence of a truncated GLUT1 did not compromise the differentiation of iPSCs into iAstros, as these cells could express several key markers representative of the astrocyte lineage. GLUT1DS-iAstros failed to express full-length GLUT1 at protein levels while showing no signs of impaired GLUT4 expression. However, GLUT1DS-iAstros showed decreased glucose uptake and lactate production compared to control-iAstros, reduced glycolysis, and mitochondrial activity as well as ATP deficit. In addition to reduced energy production, astrocytes displayed a reduced extracellular glutamate release. As previously observed, one iAstros clone (C7) showed the most severe phenotype from all groups. Our study provides an insightful view of the contribution of GLUT1 in astrocytes' energetic metabolism and raises the possible contribution of these cells in the astrocyte-neuron metabolic coupling. Our future direction is to understand better how GLUT1DS impacts astrocytes and neurons within their metabolic coupling.
    Keywords:  GLUT1; GLUT1DS; astrocytes; metabolism; stem cells
    DOI:  https://doi.org/10.1002/jcp.31523
  28. Biomolecules. 2024 Dec 30. pii: 33. [Epub ahead of print]15(1):
    VDAC1: A Key Player in the Mitochondrial Landscape of Neurodegeneration.
    Shirel Argueti-Ostrovsky, Shir Barel, Joy Kahn, Adrian Israelson.
      Voltage-Dependent Anion Channel 1 (VDAC1) is a mitochondrial outer membrane protein that plays a crucial role in regulating cellular energy metabolism and apoptosis by mediating the exchange of ions and metabolites between mitochondria and the cytosol. Mitochondrial dysfunction and oxidative stress are central features of neurodegenerative diseases. The pivotal functions of VDAC1 in controlling mitochondrial membrane permeability, regulating calcium balance, and facilitating programmed cell death pathways, position it as a key determinant in the delicate balance between neuronal viability and degeneration. Accordingly, increasing evidence suggests that VDAC1 is implicated in the pathophysiology of neurodegenerative diseases, including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and others. This review summarizes the current findings on the contribution of VDAC1 to neurodegeneration, focusing on its interactions with disease-specific proteins, such as amyloid-β, α-synuclein, and mutant SOD1. By unraveling the complex involvement of VDAC1 in neurodegenerative processes, this review highlights potential avenues for future research and drug development aimed at alleviating mitochondrial-related neurodegeneration.
    Keywords:  ALS; Alzheimer’s disease; Parkinson’s disease; VDAC1; neurodegenerative diseases
    DOI:  https://doi.org/10.3390/biom15010033
  29. Sci Transl Med. 2025 Jan 29. 17(783): eadm7580
    Early-onset sleep alterations found in patients with amyotrophic lateral sclerosis are ameliorated by orexin antagonist in mouse models.
    Simon J Guillot, Christina Lang, Marie Simonot, Daniel Beckett, Dorothée Lulé, Luisa T Balz, Antje Knehr, Geoffrey Stuart-Lopez, Pauline Vercruysse, Stéphane Dieterlé, Patrick Weydt, Johannes Dorst, Katharina Kandler, Jan Kassubek, Laura Wassermann, Caroline Rouaux, Sébastien Arthaud, Sandrine Da Cruz, Pierre-Hervé Luppi, Francesco Roselli, Albert C Ludolph, Luc Dupuis, Matei Bolborea.
      Sleep alterations have been described in several neurodegenerative diseases yet are currently poorly characterized in amyotrophic lateral sclerosis (ALS). This study investigates sleep macroarchitecture and related hypothalamic signaling disruptions in ALS. Using polysomnography, we found that both patients with ALS as well as asymptomatic C9ORF72 and SOD1 mutation carriers exhibited increased wakefulness and reduced non-rapid eye movement sleep. Increased wakefulness correlated with diminished cognitive performance in both clinical cohorts. Similar changes in sleep macroarchitecture were observed in three ALS mouse models (Sod1G86R, FusΔNLS/+, and TDP43Q331K). A single oral administration of a dual-orexin receptor antagonist or intracerebroventricular delivery of melanin-concentrating hormone (MCH) through an osmotic pump over 15 days partially normalized sleep patterns in mouse models. MCH treatment did not extend the survival of Sod1G86R mice but did decrease the loss of lumbar motor neurons. These findings suggest MCH and orexin signaling as potential targets to treat sleep alterations that arise in early stages of the disease.
    DOI:  https://doi.org/10.1126/scitranslmed.adm7580
  30. STAR Protoc. 2025 Jan 30. pii: S2666-1667(25)00015-2. [Epub ahead of print]6(1): 103609
    Protocol for assessing myelination by human iPSC-derived oligodendrocytes in Shiverer mouse ex vivo brain slice cultures.
    Themistoklis M Tsarouchas, Lida Zoupi, Anna Williams, Erin M Gibson.
      Human induced pluripotent stem cell (iPSC)-derived oligodendrocytes are a powerful tool for studying aberrant myelination in neurodegenerative and neurodevelopmental disorders; however, they often fail to myelinate in vitro. Here, we present a protocol for axonal ensheathment and perinodal segmentation using an ex vivo model. We describe steps for preparing Shiverer mouse brain slice cultures, oligodendrocyte transplantation, visualization, and analysis. This approach suits multiple culture formats, highlighting the potential for the screening of myelin-modulating drugs and compounds in a cost- and time-effective manner, while reducing animal use.
    Keywords:  Cell Differentiation; Neuroscience; Stem Cells
    DOI:  https://doi.org/10.1016/j.xpro.2025.103609
  31. Neurobiol Dis. 2025 Jan 26. pii: S0969-9961(25)00030-0. [Epub ahead of print]206 106814
    Mutations in hnRNP A1 drive neurodegeneration and alternative RNA splicing of neuronal gene targets.
    Ansalna Ansari, Patricia A Thibault, Hannah E Salapa, Joseph-Patrick W E Clarke, Michael C Levin.
      RNA binding protein dysfunction is a pathogenic feature of multiple neurological diseases, including multiple sclerosis (MS). Neurodegeneration (the loss of, or damage to neurons and axons) is the primary driver of disease progression in MS. Herein, we utilized a novel, neuron-specific model of neurodegeneration by transducing primary mouse neurons with mutant forms of the RNA binding protein heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) identified from MS patients, including one within the M9-nuclear localization sequence of hnRNP A1 (A1(P275S)) and a second in the prion-like domain of hnRNP A1 (A1(F263S)) to test the hypothesis that neuronal hnRNP A1 dysfunction drives neurodegeneration in MS. Examination of hnRNP A1 localization in neurons revealed an increase in nucleocytoplasmic mislocalization in neurons transduced with A1(P275S), but not A1(F263S). Yet, both A1(F263S) and A1(P275S) induced neurodegeneration evidenced by significant reductions in total neurite length and complexity and an increase in FluoroJade-C neuronal cell body staining. RNA sequencing and differential alternative splicing analysis of mutant-expressing neurons revealed dramatic changes in alternative RNA splicing of transcripts critical to neuronal function. Further, amyloid precursor protein (APP), a marker for neurodegeneration in MS, showed differential splicing in mutant-expressing neurons, which was confirmed in MS brains with hnRNP A1 dysfunction. Overall, we have identified that hnRNP A1 plays a complex role in neuronal function and regulation by mediating the alternative splicing of neuron-specific transcripts. When neuronal hnRNP A1 function is impaired, as in disease, resultant dysfunction propagates through multiple pathways that may influence the progression of neurodegeneration in MS.
    Keywords:  Differential splicing; In vitro MS model; Neuronal degeneration; Transcriptomics
    DOI:  https://doi.org/10.1016/j.nbd.2025.106814
  32. Acta Naturae. 2024 Oct-Dec;16(4):16(4): 73-80
    Intraventricular Administration of Exosomes from Patients with Amyotrophic Lateral Sclerosis Provokes Motor Neuron Disease in Mice.
    A V Stavrovskaya, D N Voronkov, A K Pavlova, A S Olshanskiy, B V Belugin, M V Ivanova, M N Zakharova, S N Illarioshkin.
      Amyotrophic lateral sclerosis (ALS) is a severe disease of the central nervous system (CNS) characterized by motor neuron damage leading to death from respiratory failure. The neurodegenerative process in ALS is characterized by an accumulation of aberrant proteins (TDP-43, SOD1, etc.) in CNS cells. The trans-synaptic transmission of these proteins via exosomes may be one of the mechanisms through which the pathology progresses. The aim of this work was to study the effect of an intraventricular injection of exosomes obtained from the cerebrospinal fluid (CSF) of ALS patients on the motor activity and CNS pathomorphology of mice. The exosomes were obtained from two ALS patients and a healthy donor. Exosome suspensions at high and low concentrations were injected into the lateral brain ventricles of male BALB/c mice (n = 45). Motor activity and physiological parameters were evaluated twice a month; morphological examination of the spinal cord was performed 14 months after the start of the experiment. Nine months after administration of exosomes from the ALS patients, the animals started exhibiting a pathological motor phenotype; i.e., altered locomotion with paresis of hind limbs, coordination impairment, and increasing episodes of immobility. The motor symptoms accelerated after administration of a higher concentration of exosomes. The experimental group showed a significant decrease in motor neuron density in the ventral horns of the spinal cord, a significant increase in the number of microglial cells, and microglia activation. The TDP43 protein in the control animals was localized in the nuclei of motor neurons. TDP43 mislocation with its accumulation in the cytoplasm was observed in the experimental group. Thus, the triggering effect of the exosomal proteins derived from the CSF of ALS patients in the development of a motor neuron pathology in the experimental animals was established. This confirms the pathogenetic role of exosomes in neurodegenerative progression and makes it possible to identify a new target for ALS therapy.
    Keywords:  TDP43; amyotrophic lateral sclerosis; exosomes; motor neurons; neurodegeneration
    DOI:  https://doi.org/10.32607/actanaturae.27499
  33. Biomed Pharmacother. 2025 Jan 23. pii: S0753-3322(25)00021-6. [Epub ahead of print]183 117827
    The role of mitochondrial dysfunction in Huntington's disease: Implications for therapeutic targeting.
    Deepak Chandra Joshi, Mayuri Bapu Chavan, Kajal Gurow, Madhu Gupta, Jagjit Singh Dhaliwal, Long Chiau Ming.
      Huntington's disease (HD) is a progressive, autosomal dominant neurodegenerative disorder characterized by cognitive decline, motor dysfunction, and psychiatric disturbances. A common feature of neurodegenerative disorders is mitochondrial dysfunction, which affects the brain's sensitivity to oxidative damage and its high oxygen demand. This dysfunction may plays a significant role in the pathogenesis of Huntington's disease. HD is caused by a CAG repeat expansion in the huntingtin gene, which leads to the production of a toxic mutant huntingtin (mHTT) protein. This disruption in mitochondrial function compromises energy metabolism and increases oxidative stress, resulting in mitochondrial DNA abnormalities, impaired calcium homeostasis, and altered mitochondrial dynamics. These effects ultimately may contribute to neuronal dysfunction and cell death, underscoring the importance of targeting mitochondrial function in developing therapeutic strategies for HD. This review discusses the mechanistic role of mitochondrial dysfunction in Huntington's disease. Mitochondrial dysfunction is a crucial factor in HD, making mitochondrial-targeted therapies a promising approach for treatment. We explore therapies that address bioenergy deficits, antioxidants that reduce reactive oxygen species, calcium modulators that restore calcium homeostasis, and treatments that enhance mitochondrial dynamics to rejuvenate mitochondrial function. We also highlight innovative treatment approaches such as gene editing and stem cell therapy, which offer hope for more personalized strategies. In conclusion, understanding mitochondrial dysfunction in Huntington's disease may guide potential treatment strategies. Targeting this dysfunction may help to slow disease progression and enhance the quality of life for individuals affected by Huntington's disease.
    Keywords:  Etc; Huntington's disease; Mitochondrial dysfunction; Neurodegenerative disorders; Oxidative damage
    DOI:  https://doi.org/10.1016/j.biopha.2025.117827
  34. Int J Mol Sci. 2025 Jan 13. pii: 620. [Epub ahead of print]26(2):
    Two- and Three-Dimensional In Vitro Models of Parkinson's and Alzheimer's Diseases: State-of-the-Art and Applications.
    Cristina Solana-Manrique, Ana María Sánchez-Pérez, Nuria Paricio, Silvia Muñoz-Descalzo.
      In vitro models play a pivotal role in advancing our understanding of neurodegenerative diseases (NDs) such as Parkinson's and Alzheimer's disease (PD and AD). Traditionally, 2D cell cultures have been instrumental in elucidating the cellular mechanisms underlying these diseases. Cultured cells derived from patients or animal models provide valuable insights into the pathological processes at the cellular level. However, they often lack the native tissue environment complexity, limiting their ability to fully recapitulate their features. In contrast, 3D models offer a more physiologically relevant platform by mimicking the 3D brain tissue architecture. These models can incorporate multiple cell types, including neurons, astrocytes, and microglia, creating a microenvironment that closely resembles the brain's complexity. Bioengineering approaches allow researchers to better replicate cell-cell interactions, neuronal connectivity, and disease-related phenotypes. Both 2D and 3D models have their advantages and limitations. While 2D cultures provide simplicity and scalability for high-throughput screening and basic processes, 3D models offer enhanced physiological relevance and better replicate disease phenotypes. Integrating findings from both model systems can provide a better understanding of NDs, ultimately aiding in the development of novel therapeutic strategies. Here, we review existing 2D and 3D in vitro models for the study of PD and AD.
    Keywords:  Alzheimer’s disease; Parkinson’s disease; engineering-based 3D models; iPSCs; immortalised cell lines; in vitro models; organoids
    DOI:  https://doi.org/10.3390/ijms26020620
  35. CNS Neurosci Ther. 2025 Jan;31(1): e70240
    Morphological Characteristics and Extracellular Matrix Abnormalities in Astrocytes Derived From iPSCs of Children With Alexander Disease.
    Huan Yi, Jie Zhang, Kai Gao, Wei Yan, Hongyuan Chu, Junjiao Zhang, Fan Zhang, Yuwu Jiang, Jingmin Wang, Ye Wu.
       AIMS: Alexander disease (AxD) is a leukodystrophy caused by mutations in the astrocytic filament gene GFAP. There are currently no effective treatments for AxD. Previous studies have rarely established AxD models with the patient's original GFAP mutations. In this study, we aimed to explore the morphological and transcriptomic characteristics of GFAP-mutant astrocytes via induced pluripotent stem cell (iPSC) models of AxD.
    METHODS: Fibroblasts from three AxD children were reprogrammed into iPSCs. Wild-type (WT) and AxD-iPSCs were differentiated into astrocytes. We compared the morphological and transcriptomic differences between WT- and AxD iPSC-derived astrocytes.
    RESULTS: Astrocytes induced from AxD-derived iPSCs exhibited the Rosenthal fibers (RFs), the main pathological phenotype of AxD. Compared with WT astrocytes, AxD astrocytes had shorter processes, more branches, and larger cell bodies. Transcriptomic analysis revealed that extracellular matrix (ECM) components, particularly chondroitin sulfate proteoglycans (CSPGs), were upregulated, and ECM-degrading enzymes were generally downregulated. These changes may lead to abnormalities in neurons and myelination.
    CONCLUSIONS: We explored the morphological characteristics of AxD astrocytes via iPSC models and revealed the ECM, previously unexplored for AxD, may be an important new pathogenic mechanism of this disease.
    Keywords:  Alexander disease; GFAP; Rosenthal fibers; extracellular matrix; induced pluripotent stem cells
    DOI:  https://doi.org/10.1111/cns.70240
  36. Biomedicines. 2025 Jan 07. pii: 122. [Epub ahead of print]13(1):
    Novel Poly-Arginine Peptide R18D Reduces α-Synuclein Aggregation and Uptake of α-Synuclein Seeds in Cortical Neurons.
    Emma C Robinson, Anastazja M Gorecki, Samuel R Pesce, Vaishali Bagda, Ryan S Anderton, Bruno P Meloni.
       BACKGROUND/OBJECTIVES: The role of α-synuclein (α-syn) pathology in Parkinson's disease (PD) is well established; however, effective therapies remain elusive. Two mechanisms central to PD neurodegeneration are the intracellular aggregation of misfolded α-syn and the uptake of α-syn aggregates into neurons. Cationic arginine-rich peptides (CARPs) are an emerging class of molecule with multiple neuroprotective mechanisms of action, including protein stabilisation. This study characterised both intracellular α-syn aggregation and α-syn uptake in cortical neurons in vitro. Thereafter, this study examined the therapeutic potential of the neuroprotective CARP, R18D (18-mer of D-arginine), to prevent the aforementioned PD pathogenic processes through a cell-free thioflavin-T (ThT) assay and in cortical neurons.
    METHODS: To induce intracellular α-syn aggregation, rat primary cortical neurons were exposed to α-syn seed (0.14 μM) for 2 h to allow uptake of the protein, followed by R18D treatment (0.0625, 0.125, 0.25, 0.5 μM), and a subsequent measurement of α-syn aggregates 48 h later using a homogenous time-resolved fluorescence (HTRF) assay. To assess neuronal uptake, α-syn seeds were covalently labelled with an Alexa-Fluor 488 fluorescent tag, pre-incubated with R18D (0.125, 0.25, 0.5 μM), and then exposed to cortical neurons for 24 h and assessed via confocal microscopy.
    RESULTS: It was demonstrated that R18D significantly reduced both intracellular α-syn aggregation and α-syn seed uptake in neurons by 37.8% and 77.7%, respectively. Also, R18D reduced the aggregation of α-syn monomers in the cell-free assay.
    CONCLUSIONS: These findings highlight the therapeutic potential of R18D to inhibit key α-syn pathological processes and PD progression.
    Keywords:  Parkinson’s disease; R18D; cationic arginine-rich peptides; primary cortical neurons; α-synuclein; α-synuclein seeds
    DOI:  https://doi.org/10.3390/biomedicines13010122
  37. Biomedicines. 2024 Dec 27. pii: 35. [Epub ahead of print]13(1):
    Stem Cell Therapy for the Treatment of Amyotrophic Lateral Sclerosis: Comparison of the Efficacy of Mesenchymal Stem Cells, Neural Stem Cells, and Induced Pluripotent Stem Cells.
    Lauren Frawley, Noam Tomer Taylor, Olivia Sivills, Ella McPhillamy, Timothy Duy To, Yibo Wu, Beek Yoke Chin, Chiew Yen Wong.
       BACKGROUND/OBJECTIVES: Amyotrophic lateral sclerosis (ALS), or Lou Gehrig's disease, is a debilitating, incurable neurodegenerative disorder characterised by motor neuron death in the spinal cord, brainstem, and motor cortex. With an incidence rate of about 4.42 cases per 100,000 people annually, ALS severely impacts motor function and quality of life, causing progressive muscle atrophy, spasticity, paralysis, and eventually death. The cause of ALS is largely unknown, with 90% of cases being sporadic and 10% familial. Current research targets molecular mechanisms of inflammation, excitotoxicity, aggregation-prone proteins, and proteinopathy.
    METHODS: This review evaluates the efficacy of three stem cell types in ALS treatment: mesenchymal stem cells (MSCs), neural stem cells (NSCs), and induced pluripotent stem cells (iPSCs).
    RESULTS: MSCs, derived from various tissues, show neuroprotective and regenerative qualities, with clinical trials suggesting potential benefits but limited by small sample sizes and non-randomised designs. NSCs, isolated from the fetal spinal cord or brain, demonstrate promise in animal models but face functional integration and ethical challenges. iPSCs, created by reprogramming patient-specific somatic cells, offer a novel approach by potentially replacing or supporting neurons. iPSC therapy addresses ethical issues related to embryonic stem cells but encounters challenges regarding genotoxicity and epigenetic irregularities, somatic cell sources, privacy concerns, the need for extensive clinical trials, and high reprogramming costs.
    CONCLUSIONS: This research is significant for advancing ALS treatment beyond symptomatic relief and modest survival extensions to actively modifying disease progression and improving patient outcomes. Successful stem cell therapies could lead to new ALS treatments, slowing motor function loss and reducing symptom severity.
    Keywords:  amyotrophic lateral sclerosis (ALS); induced pluripotent stem cells (iPSCs); mesenchymal stem cells (MSCs); neural stem cells (NSCs); regenerative medicine; stem cell therapy (SCT)
    DOI:  https://doi.org/10.3390/biomedicines13010035
  38. Bio Protoc. 2025 Jan 20. 15(2): e5169
    Primary Neuronal Culture and Transient Transfection.
    Shun-Cheng Tseng, Peng-Tzu Chen, Eric Hwang.
      Primary neuronal culture and transient transfection offer a pair of crucial tools for neuroscience research, providing a controlled environment to study the behavior, function, and interactions of neurons in vitro. These cultures can be used to investigate fundamental aspects of neuronal development and plasticity, as well as disease mechanisms. There are numerous methods of transient transfection, such as electroporation, calcium phosphate precipitation, or cationic lipid transfection. In this protocol, we used electroporation for neurons immediately before plating and cationic lipid transfection for neurons that have been cultured for a few days in vitro. In our experience, the transfection efficiency of electroporation can be as high as 30%, and cationic lipid transfection has an efficiency of 1%-2%. While cationic lipid transfection has much lower efficiency than electroporation, it does offer the advantage of a higher expression level. Therefore, these transfection methods are suitable for different stages of neurons and different expression requirements. Key features • Culture of primary neurons from the CNS. • Electroporation for freshly isolated neurons in suspension. • Cationic lipid transfection for adherent neurons.
    Keywords:  Adherent neuron transient transfection; Neurite outgrowth; Neuronal isolation; Neuronal morphology; Suspension neuron transient transfection
    DOI:  https://doi.org/10.21769/BioProtoc.5169
  39. J Vis Exp. 2025 Jan 10.
    Quantitative Analysis of Mitochondria-Associated Endoplasmic Reticulum Membrane (MAM) Stabilization in a Neural Model of Alzheimer's Disease (AD).
    Jacob C Zellmer, Selene Lomoio, Rudolph E Tanzi, Raja Bhattacharyya.
      A method to quantitate the stabilization of Mitochondria-Associated endoplasmic reticulum Membranes (MAMs) in a 3-dimensional (3D) neural model of Alzheimer's disease (AD) is presented here. To begin, fresh human neuro progenitor ReN cells expressing β-amyloid precursor protein (APP) containing familial Alzheimer's disease (FAD) or naïve ReN cells are grown in thin (1:100) Matrigel-coated tissue culture plates. After the cells reach confluency, these are electroporated with expression plasmids encoding red fluorescence protein (RFP)-conjugated mitochondria-binding sequence of AKAP1(34-63) (Mito-RFP) that detects mitochondria or constitutive MAM stabilizers MAM 1X or MAM 9X that stabilize tight (6 nm ± 1 nm gap width) or loose (24 nm ± 3 nm gap width) MAMs, respectively. After 16-24 h, the cells are harvested and enriched by a fluorescence-activated cell sorter (FACS). An equal number of FACS-enriched cells are seeded in the 3-dimensional matrix (1:1 Matrigel) and allowed to differentiate into mature neurons for 10 days. Live cell images of the 10-day differentiated cells expressing the RFP-conjugated MAM stabilizers are captured under a fluorescent microscope equipped with a live-cell imaging culture chamber maintaining the CO2 (5%), temperature (37 °C), and humidity (~90%). Toward this end, we performed live-cell imaging and kymographic analyses to measure the motility of free mitochondria labeled with Mito-RFP or ER-bound mitochondria of tight or loose gap widths stabilized by MAM 1X or MAM 9X, respectively, in the most extended neuronal process of each ReN GA neuron which is at least 500 nm long, considering these as axons.
    DOI:  https://doi.org/10.3791/66129
  40. Nat Commun. 2025 Jan 27. 16(1): 1068
    Splicing accuracy varies across human introns, tissues, age and disease.
    S García-Ruiz, D Zhang, E K Gustavsson, G Rocamora-Perez, M Grant-Peters, A Fairbrother-Browne, R H Reynolds, J W Brenton, A L Gil-Martínez, Z Chen, D C Rio, J A Botia, S Guelfi, L Collado-Torres, M Ryten.
      Alternative splicing impacts most multi-exonic human genes. Inaccuracies during this process may have an important role in ageing and disease. Here, we investigate splicing accuracy using RNA-sequencing data from >14k control samples and 40 human body sites, focusing on split reads partially mapping to known transcripts in annotation. We show that splicing inaccuracies occur at different rates across introns and tissues and are affected by the abundance of core components of the spliceosome assembly and its regulators. We find that age is positively correlated with a global decline in splicing fidelity, mostly affecting genes implicated in neurodegenerative diseases. We find support for the latter by observing a genome-wide increase in splicing inaccuracies in samples affected with Alzheimer's disease as compared to neurologically normal individuals. In this work, we provide an in-depth characterisation of splicing accuracy, with implications for our understanding of the role of inaccuracies in ageing and neurodegenerative disorders.
    DOI:  https://doi.org/10.1038/s41467-024-55607-x
  41. PLoS Biol. 2025 Jan;23(1): e3002998
    The ubiquitin-conjugating enzyme UBE2D maintains a youthful proteome and ensures protein quality control during aging by sustaining proteasome activity.
    Liam C Hunt, Michelle Curley, Kudzai Nyamkondiwa, Anna Stephan, Jianqin Jiao, Kanisha Kavdia, Vishwajeeth R Pagala, Junmin Peng, Fabio Demontis.
      Ubiquitin-conjugating enzymes (E2s) are key for protein turnover and quality control via ubiquitination. Some E2s also physically interact with the proteasome, but it remains undetermined which E2s maintain proteostasis during aging. Here, we find that E2s have diverse roles in handling a model aggregation-prone protein (huntingtin-polyQ) in the Drosophila retina: while some E2s mediate aggregate assembly, UBE2D/effete (eff) and other E2s are required for huntingtin-polyQ degradation. UBE2D/eff is key for proteostasis also in skeletal muscle: eff protein levels decline with aging, and muscle-specific eff knockdown causes an accelerated buildup in insoluble poly-ubiquitinated proteins (which progressively accumulate with aging) and shortens lifespan. Mechanistically, UBE2D/eff is necessary to maintain optimal proteasome function: UBE2D/eff knockdown reduces the proteolytic activity of the proteasome, and this is rescued by transgenic expression of human UBE2D2, an eff homolog. Likewise, human UBE2D2 partially rescues the lifespan and proteostasis deficits caused by muscle-specific effRNAi and re-establishes the physiological levels of effRNAi-regulated proteins. Interestingly, UBE2D/eff knockdown in young age reproduces part of the proteomic changes that normally occur in old muscles, suggesting that the decrease in UBE2D/eff protein levels that occurs with aging contributes to reshaping the composition of the muscle proteome. However, some of the proteins that are concertedly up-regulated by aging and effRNAi are proteostasis regulators (e.g., chaperones and Pomp) that are transcriptionally induced presumably as part of an adaptive stress response to the loss of proteostasis. Altogether, these findings indicate that UBE2D/eff is a key E2 ubiquitin-conjugating enzyme that ensures protein quality control and helps maintain a youthful proteome composition during aging.
    DOI:  https://doi.org/10.1371/journal.pbio.3002998
  42. Protein Sci. 2025 Feb;34(2): e70008
    Super-resolution imaging of proteins inside live mammalian cells with mLIVE-PAINT.
    Haresh Bhaskar, Zoe Gidden, Gurvir Virdi, Dirk-Jan Kleinjan, Susan J Rosser, Sonia Gandhi, Lynne Regan, Mathew H Horrocks.
      Super-resolution microscopy has revolutionized biological imaging, enabling the visualization of structures at the nanometer length scale. Its application in live cells, however, has remained challenging. To address this, we adapted LIVE-PAINT, an approach we established in yeast, for application in live mammalian cells. Using the 101A/101B coiled-coil peptide pair as a peptide-based targeting system, we successfully demonstrate the super-resolution imaging of two distinct proteins in mammalian cells, one localized in the nucleus, and the second in the cytoplasm. This study highlights the versatility of LIVE-PAINT, suggesting its potential for live-cell super-resolution imaging across a range of protein targets in mammalian cells. We name the mammalian cell version of our original method mLIVE-PAINT.
    Keywords:  dynamics; mitochondria; nucleus; peptide–peptide interactions; single‐molecule; super‐resolution microscopy
    DOI:  https://doi.org/10.1002/pro.70008
  43. Nat Commun. 2025 Jan 24. 16(1): 978
    SLC25A38 is required for mitochondrial pyridoxal 5'-phosphate (PLP) accumulation.
    Izabella A Pena, Jeffrey S Shi, Sarah M Chang, Jason Yang, Samuel Block, Charles H Adelmann, Heather R Keys, Preston Ge, Shveta Bathla, Isabella H Witham, Grzegorz Sienski, Angus C Nairn, David M Sabatini, Caroline A Lewis, Nora Kory, Matthew G Vander Heiden, Myriam Heiman.
      Many essential proteins require pyridoxal 5'-phosphate, the active form of vitamin B6, as a cofactor for their activity. These include enzymes important for amino acid metabolism, one-carbon metabolism, polyamine synthesis, erythropoiesis, and neurotransmitter metabolism. A third of all mammalian pyridoxal 5'-phosphate-dependent enzymes are localized in the mitochondria; however, the molecular machinery involved in the regulation of mitochondrial pyridoxal 5'-phosphate levels in mammals remains unknown. In this study, we used a genome-wide CRISPR interference screen in erythroleukemia cells and organellar metabolomics to identify the mitochondrial inner membrane protein SLC25A38 as a regulator of mitochondrial pyridoxal 5'-phosphate. Loss of SLC25A38 causes depletion of mitochondrial, but not cellular, pyridoxal 5'-phosphate, and impairs cellular proliferation under both physiological and low vitamin B6 conditions. Metabolic changes associated with SLC25A38 loss suggest impaired mitochondrial pyridoxal 5'-phosphate-dependent enzymatic reactions, including serine to glycine conversion catalyzed by serine hydroxymethyltransferase-2 as well as ornithine aminotransferase. The proliferation defect of SLC25A38-null K562 cells in physiological and low vitamin B6 media can be explained by the loss of serine hydroxymethyltransferase-2-dependent production of one-carbon units and downstream de novo nucleotide synthesis. Our work points to a role for SLC25A38 in mitochondrial pyridoxal 5'-phosphate accumulation and provides insights into the pathology of congenital sideroblastic anemia.
    DOI:  https://doi.org/10.1038/s41467-025-56130-3
  44. Eur J Neurosci. 2025 Jan;61(1): e16659
    Oxyglutamate Carrier Alleviates Cerebral Ischaemia-Reperfusion Injury by Regulating Mitochondrial Function.
    Wenhao Liu, Xin Liu, Min Liu, Rui Zhao, Zhiyuan Zhao, Jingrui Xiao, Dongdong Wan, Qi Wan, Rui Xu.
      Mitochondrial dysfunction has been reported to participate in the pathophysiological processes of cerebral ischaemia-reperfusion injury, which include reduced energy homeostasis, increased generation of oxidative stress species (ROS) and the release of apoptotic factors. Oxyglutamate carrier (OGC) is an important carrier protein on the inner mitochondrial membrane that can transport metabolites from the cytoplasm to the mitochondria. The role of OGC in cerebral ischaemia-reperfusion injury (I/R) remains unknown. In this study, we found that the expression of OGC was significantly upregulated after cerebral ischaemia-reperfusion injury. Inhibiting OGC with phenylsuccinic acid (PSA) increased neuronal death after oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro. Mechanistically, OGC was localized in mitochondria and could facilitate the transport of glutathione from the cytoplasm to the mitochondria to reduce ROS levels and increase ATP production after OGD/R. In addition, in vivo inhibition of OGC exacerbated brain infarction, and GSH supplementation alleviated brain infarction resulting from OGC inhibition. This study revealed the role of OGC in alleviating brain damage by regulating mitochondrial GSH transport to alleviate mitochondrial function after cerebral ischaemia-reperfusion injury, which may provide a target for alleviating ischaemic brain injury.
    Keywords:  OGC; ROS; ischaemia–reperfusion injury; mitochondria; neuron
    DOI:  https://doi.org/10.1111/ejn.16659
  45. Cell Insight. 2025 Apr;4(2): 100222
    Inflammatory signatures of microglia in hypercortisolemia-related depression.
    Yanxiang Zhao, Yingying Huang, Zhangyuzi Deng, Ying Cao, Jing Yang.
      
    DOI:  https://doi.org/10.1016/j.cellin.2024.100222
  46. Curr Top Med Chem. 2025 Jan 27.
    Roles of C/EBPβ/AEP in Neurodegenerative Diseases.
    Jing Guo, Xin-Yi Liu, Sha-Sha Yang, Qiang Li, Yang Duan, Shan-Shan Zhu, Ke Zhou, Yi-Zhi Yan, Peng Zeng.
      In recent years, an increasing number of studies have shown that increased activation of aspartic endopeptidases (AEPs) is a common symptom in neurodegenerative diseases (NDDs). AEP cleaves amyloid precursor protein (APP), tau (microtubule-associated protein tau), α- synuclein (α-syn), SET (a 39-KDa phosphoprotein widely expressed in various tissues and localizes predominantly in the nucleus), and TAR DNA-binding protein 43 (TDP-43), and promotes their aggregation, contributing to Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease, multiple sclerosis (MS), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD) pathogenesis. Abundant evidence supports the notion that CCAAT/enhancer-binding protein β (C/EBPβ)/AEP may play an important role in NDDs. Developing its small molecule inhibitors is a promising treatment of NDDs. However, current research suggests that the pathophysiological mechanism of the C/EBPβ/AEP pathway is very complex in NDDs. This review summarizes the structure of C/EBPβ and AEP, their major physiological functions, potential pathogenesis, their small molecule inhibitors, and how C/EBPβ/AEP offers a novel pathway for the treatment of NDDs.
    Keywords:  C/EBPβ; Neurodegenerative diseases; asparagine endopeptidase; pathogenesis; small molecule inhibitors.
    DOI:  https://doi.org/10.2174/0115680266357822250119172351
  47. Biol Open. 2025 Feb 15. pii: BIO061601. [Epub ahead of print]14(2):
    Increased expression of the small lysosomal gene SVIP in the Drosophila gut suppresses pathophysiological features associated with a high-fat diet.
    Brennan M Mercola, Tatiana V Villalobos, Jocelyn E Wood, Ankita Basu, Alyssa E Johnson.
      Lysosomes are digestive organelles that are crucial for nutrient sensing and metabolism. Lysosome impairment is linked to a broad spectrum of metabolic disorders, underscoring their importance to human health. Thus, lysosomes are an attractive target for metabolic disease therapies. In previous work, we discovered a novel class of tubular lysosomes that are morphologically and functionally distinct from traditionally described vesicular lysosomes. Tubular lysosomes are present in multiple tissues, are broadly conserved from invertebrates to mammals, are more proficient at degrading autophagic cargo than vesicular lysosomes, and delay signs of tissue aging when induced ectopically. Thus, triggering tubular lysosome formation presents one mechanism to increase lysosome activity and, notably, overproduction of the small lysosomal protein, SVIP, is a robust genetic strategy for triggering lysosomal tubulation on demand. In this study, we examine whether SVIP overexpression in the fly gut can suppress pathophysiological phenotypes associated with an obesogenic high-fat diet. Indeed, our results indicate that increasing SVIP expression in the fly gut reduces lipid accumulation, suppresses body mass increase, and improves survival in flies fed a high-fat diet. Collectively, these data hint that increasing lysosomal activity through induction of tubular lysosomal networks, could be one strategy to combat obesity-related pathologies.
    Keywords:   Drosophila ; Autophagy; High-fat diet; Lysosomes; Obesity; SVIP
    DOI:  https://doi.org/10.1242/bio.061601
  48. Int J Mol Sci. 2025 Jan 10. pii: 549. [Epub ahead of print]26(2):
    The Type III Intermediate Filament Protein Peripherin Regulates Lysosomal Degradation Activity and Autophagy.
    Roberta Romano, Paola Cordella, Cecilia Bucci.
      Peripherin belongs to heterogeneous class III of intermediate filaments, and it is the only intermediate filament protein selectively expressed in the neurons of the peripheral nervous system. It has been previously discovered that peripherin interacts with proteins important for the endo-lysosomal system and for the transport to late endosomes and lysosomes, such as RAB7A and AP-3, although little is known about its role in the endocytic pathway. Here, we show that peripherin silencing affects lysosomal abundance but also positioning, causing the redistribution of lysosomes from the perinuclear area to the cell periphery. Moreover, peripherin silencing affects lysosomal activity, inhibiting EGFR degradation and the degradation of a fluorogenic substrate for proteases. Furthermore, we demonstrate that peripherin silencing affects lysosomal biogenesis by reducing the TFEB and TFE3 contents. Finally, in peripherin-depleted cells, the autophagic flux is strongly inhibited. Therefore, these data indicate that peripherin has an important role in regulating lysosomal biogenesis, and positioning and functions of lysosomes, affecting both the endocytic and autophagic pathways. Considering that peripherin is the most abundant intermediate filament protein of peripheral neurons, its dysregulation, affecting its functions, could be involved in the onset of several neurodegenerative diseases of the peripheral nervous system characterized by alterations in the endocytic and/or autophagic pathways.
    Keywords:  autophagy; cytoskeleton; intermediate filaments; lysosome; peripherin
    DOI:  https://doi.org/10.3390/ijms26020549
  49. Front Mol Neurosci. 2024 ;17 1527013
    Novel strategies targeting mitochondria-lysosome contact sites for the treatment of neurological diseases.
    Yinyin Xie, Wenlin Sun, Aoya Han, Xinru Zhou, Shijie Zhang, Changchang Shen, Yi Xie, Cui Wang, Nanchang Xie.
      Mitochondria and lysosomes are critical for neuronal homeostasis, as highlighted by their dysfunction in various neurological diseases. Recent studies have identified dynamic membrane contact sites between mitochondria and lysosomes, independent of mitophagy and the lysosomal degradation of mitochondrial-derived vesicles (MDVs), allowing bidirectional crosstalk between these cell compartments, the dynamic regulation of organelle networks, and substance exchanges. Emerging evidence suggests that abnormalities in mitochondria-lysosome contact sites (MLCSs) contribute to neurological diseases, including Parkinson's disease, Charcot-Marie-Tooth (CMT) disease, lysosomal storage diseases, and epilepsy. This article reviews recent research advances regarding the tethering processes, regulation, and function of MLCSs and their role in neurological diseases.
    Keywords:  lysosomal dynamics; mitochondria-lysosome contact sites; mitochondrial network; mitophagy; neurological diseases; substance exchanges
    DOI:  https://doi.org/10.3389/fnmol.2024.1527013
  50. Mol Cell Neurosci. 2025 Jan 23. pii: S1044-7431(25)00002-8. [Epub ahead of print]132 103992
    Mitochondrial fission and fusion in neurodegenerative diseases:Ca2+ signalling.
    Xuan Liu, Tianjiao Li, Xinya Tu, Mengying Xu, Jianwu Wang.
      Neurodegenerative diseases (NDs) are a group of disorders characterized by the progressive loss of neuronal structure and function. The pathogenesis is intricate and involves a network of interactions among multiple causes and systems. Mitochondria and Ca2+ signaling have long been considered to play important roles in the development of various NDs. Mitochondrial fission and fusion dynamics are important processes of mitochondrial quality control, ensuring the stability of mitochondrial structure and function. Mitochondrial fission and fusion imbalance and Ca2+ signaling disorders can aggravate the disease progression of NDs. In this review, we explore the relationship between mitochondrial dynamics and Ca2+ signaling in AD, PD, ALS, and HD, focusing on the roles of key regulatory proteins (Drp1, Fis1, Mfn1/2, and Opa1) and the association structures between mitochondria and the endoplasmic reticulum (MERCs/MAMs). We provide a detailed analysis of their involvement in the pathogenesis of these four NDs. By integrating these mechanisms, we aim to clarify their contributions to disease progression and offer insights into the development of therapeutic strategies that target mitochondrial dynamics and Ca2+ signaling. We also examine the progress in drug research targeting these pathways, highlighting their potential as therapeutic targets in the treatment of NDs.
    Keywords:  Ca(2+) signaling; Mitochondrial fusion and fission; Mitochondrial-associated membranes (MAMs); Mitochondrial–ER contact sites (MERCs); Neurodegenerative diseases; Potential drugs
    DOI:  https://doi.org/10.1016/j.mcn.2025.103992
  51. ASN Neuro. 2025 ;17(1): 2443442
    New Atg9 Phosphorylation Sites Regulate Autophagic Trafficking in Glia.
    Linfang Wang, Shuanglong Yi, Shiping Zhang, Yu-Ting Tsai, Yi-Hsuan Cheng, Yu-Tung Lin, Chia-Ching Lin, Yi-Hua Lee, Honglei Wang, Margaret S Ho.
      We previously identified a role for dAuxilin (dAux), the fly homolog of Cyclin G-associated kinase, in glial autophagy contributing to Parkinson's disease (PD). To further dissect the mechanism, we present evidence here that lack of glial dAux enhanced the phosphorylation of the autophagy-related protein Atg9 at two newly identified threonine residues, T62 and T69. The enhanced Atg9 phosphorylation in the absence of dAux promotes autophagosome formation and Atg9 trafficking to the autophagosomes in glia. Whereas the expression of the non-phosphorylatable Atg9 variants suppresses the lack of dAux-induced increase in both autophagosome formation and Atg9 trafficking to autophagosome, the expression of the phosphomimetic Atg9 variants restores the lack of Atg1-induced decrease in both events. In relation to pathophysiology, Atg9 phosphorylation at T62 and T69 contributes to dopaminergic neurodegeneration and locomotor dysfunction in a Drosophila PD model. Notably, increased expression of the master autophagy regulator Atg1 promotes dAux-Atg9 interaction. Thus, we have identified a dAux-Atg1-Atg9 axis relaying signals through the Atg9 phosphorylation at T62 and T69; these findings further elaborate the mechanism of dAux regulating glial autophagy and highlight the significance of protein degradation pathway in glia contributing to PD.
    Keywords:  Atg1; Atg9; Parkinson’s disease; dAuxilin; glia
    DOI:  https://doi.org/10.1080/17590914.2024.2443442
  52. Electronics (Basel). 2024 Dec 02. pii: 4985. [Epub ahead of print]13(24):
    Data-Driven Maturity Level Evaluation for Cardiomyocytes Derived from Human Pluripotent Stem Cells (Invited Paper).
    Yan Hong, Xueqing Huang, Fang Li, Siqi Huang, Qibiao Weng, Diego Fraidenraich, Ioana Voiculescu.
      Cardiovascular disease is a leading cause of death worldwide. The differentiation of human pluripotent stem cells (hPSCs) into functional cardiomyocytes offers significant potential for disease modeling and cell-based cardiac therapies. However, hPSC-derived cardiomyocytes (hPSC-CMs) remain largely immature, limiting their experimental and clinical applications. A critical challenge in current in vitro culture systems is the absence of standardized metrics to quantify maturity. This study presents a data-driven pipeline to quantify hPSC-CM maturity using gene expression data across various stages of cardiac development. We determined that culture time serves as a feasible proxy for maturity. To improve prediction accuracy, machine learning algorithms were employed to identify heart-related genes whose expression strongly correlates with culture time. Our results reduced the average discrepancy between predicted and observed culture time to 4.461 days and CASQ2 (Calsequestrin 2), a gene involved in calcium ion storage and transport, was identified as the most critical cardiac gene associated with culture duration. This novel framework for maturity assessment moves beyond traditional qualitative methods, providing deeper insights into hPSC-CM maturation dynamics. It establishes a foundation for developing advanced lab-on-chip devices capable of real-time maturity monitoring and adaptive stimulus selection, paving the way for improved maturation strategies and broader experimental/clinical applications.
    Keywords:  cardiac gene selection; cardiovascular diseases; culture time prediction; gene expression; hPSC-CM maturity
    DOI:  https://doi.org/10.3390/electronics13244985
  53. J Cell Biol. 2025 Mar 03. pii: e202311082. [Epub ahead of print]224(3):
    FAM43A coordinates mtDNA replication and mitochondrial biogenesis in response to mtDNA depletion.
    Alva G Sainz, Gladys R Rojas, Alexandra G Moyzis, Matthew P Donnelly, Kailash C Mangalhara, Melissa A Johnson, Pau B Esparza-Moltó, Kym J Grae, Reuben J Shaw, Gerald S Shadel.
      Mitochondrial retrograde signaling (MRS) pathways relay the functional status of mitochondria to elicit homeostatic or adaptive changes in nuclear gene expression. Budding yeast have "intergenomic signaling" pathways that sense the amount of mitochondrial DNA (mtDNA) independently of oxidative phosphorylation (OXPHOS), the primary function of genes encoded by mtDNA. However, MRS pathways that sense the amount of mtDNA in mammalian cells remain poorly understood. We found that mtDNA-depleted IMR90 cells can sustain OXPHOS for a significant amount of time, providing a robust model system to interrogate human intergenomic signaling. We identified FAM43A, a largely uncharacterized protein, as a CHK2-dependent early responder to mtDNA depletion. Depletion of FAM43A activates a mitochondrial biogenesis program, resulting in an increase in mitochondrial mass and mtDNA copy number via CHK2-mediated upregulation of the p53R2 form of ribonucleotide reductase. We propose that FAM43A performs a checkpoint-like function to limit mitochondrial biogenesis and turnover under conditions of mtDNA depletion or replication stress.
    DOI:  https://doi.org/10.1083/jcb.202311082
  54. Curr Protein Pept Sci. 2025 Jan 27.
    Chaperones as Potential Pharmacological Targets for Treating Protein Aggregation Illness.
    Shikha Rani, Minkal Tuteja.
      The three-dimensional structure of proteins, achieved through the folding of the nascent polypeptide chain in vivo, is largely facilitated by molecular chaperones, which are crucial for determining protein functionality. In addition to aiding in the folding process, chaperones target misfolded proteins for degradation, acting as a quality control system within the cell. Defective protein folding has been implicated in a wide range of clinical conditions, including neurodegenerative and metabolic disorders. It is now well understood that the pathogenesis of neurodegenerative diseases such as Parkinson's disease, Alzheimer's disease, Huntington's disease, Amyotrophic Lateral Sclerosis, and Creutzfeldt-Jakob disease shares a common mechanism: the accumulation of misfolded proteins, which aggregate and become toxic to cells. Among the family of molecular chaperones, Heat Shock Proteins (HSPs) are highly expressed in response to cellular stress and play a pivotal role in preventing protein aggregation. Specific chaperones, particularly HSPs, are now recognized as critical in halting the accumulation and aggregation of misfolded proteins in these conditions. Consequently, these chaperones are increasingly considered promising pharmacological targets for the treatment of protein aggregation-related diseases. This review highlights research exploring the potential roles of specific molecular chaperones in disorders characterized by the accumulation of misfolded proteins.
    Keywords:  Protein folding; aggregates; and therapeutic targets; chaperone therapy; conformational disorders; misfolding
    DOI:  https://doi.org/10.2174/0113892037338028241230092414
  55. Front Toxicol. 2024 ;6 1523387
    A primary rat neuron-astrocyte-microglia tri-culture model for studying mechanisms of neurotoxicity.
    Jessie R Badley, Aishwarya Bhusal, Pamela J Lein.
      Primary cell cultures from rodent brain are widely used to investigate molecular and cellular mechanisms of neurotoxicity. To date, however, it has been challenging to reliably culture endogenous microglia in dissociated mixed cultures. This is a significant limitation of most in vitro neural cell models given the growing awareness of the importance of interactions between neurons, astrocytes and microglia in defining responses to neurotoxic exposures. We recently developed a tri-culture model consisting of neurons, astrocytes and microglia dissociated from the developing rat neocortex and demonstrated that this tri-culture model more faithfully mimics in vivo neuroinflammatory responses then standard neuron-only or neuron-astrocyte co-cultures. Here, we describe our protocol for generating tri-cultures of rat cortical neurons, astrocytes and microglia in which all 3 cell types can be maintained for up to 1 month in culture at the same relative ratio observed in the developing rat neocortex. We also discuss applications of this model for neurotoxicity testing, as well as the potential of this model to fill a current gap for assessing neuroinflammation in the in vitro testing battery for developmental neurotoxicity.
    Keywords:  cortical neurons; in vitro model; neuroinflammation; neurotoxicity; rat
    DOI:  https://doi.org/10.3389/ftox.2024.1523387
  56. Neurobiol Dis. 2025 Jan 22. pii: S0969-9961(25)00026-9. [Epub ahead of print]206 106810
    Huntingtin plays an essential role in the adult hippocampus.
    Jessica C Barron, Laura J Dawson, Samantha J Carew, Mackenzie C Grace, Kelsie A Senior, Katelyn C Ryan, Firoozeh Nafar, Craig S Moore, Jacqueline Blundell, Matthew P Parsons.
      The consequences of non-pathogenic huntingtin (HTT) reduction in the mature brain are of substantial importance as clinical trials for numerous HTT-lowering therapies are underway; many of which are non-selective in that they reduce both mutant and wild type protein variants. In this study, we injected CaMKII-promoted AAV-Cre directly into the hippocampus of adult HTT floxed mice to explore the role of wild-type huntingtin (wtHTT) in adult hippocampal pyramidal neurons and the broader implications of its loss. Our findings reveal that wtHTT depletion results in profound macroscopic morphological abnormalities in hippocampal structure, accompanied by significant reactive gliosis. At the synaptic level, we identified a marked reduction in presynaptic terminals 1-2 months following wtHTT loss; this was contrasted by an increased density of postsynaptic mushroom spines and larger amplitudes of spontaneous excitatory postsynaptic currents, indicative of disrupted synaptic homeostasis. Furthermore, intrinsic neuronal excitability was significantly diminished in CA1 pyramidal neurons lacking wtHTT, and we observed a complete loss of NMDA receptor-dependent long-term potentiation. Unexpectedly, synapse density returned to control levels 6-8 months following wtHTT loss, despite the ongoing presence of macroscopic morphological abnormalities, altered anxiety-related behaviors and clear impairments in spatial learning and memory. Overall, these findings uncover a previously unrecognized role of wtHTT as a critical regulator of hippocampal function in the mature brain, and highlight the hippocampus as a potentially vulnerable region to the adverse effects of non-selective HTT reduction.
    Keywords:  Hippocampus; Huntingtin; Huntington's disease; Learning and memory; Neurobiology; Neuroinflammation; Synaptic plasticity
    DOI:  https://doi.org/10.1016/j.nbd.2025.106810
  57. bioRxiv. 2025 Jan 18. pii: 2025.01.17.633664. [Epub ahead of print]
    Hypoxia-inducible factor 1 protects neurons from Sarm1-mediated neurodegeneration.
    Paul Meraner, Adel Avetisyan, Kevin Swift, Ya-Chen Cheng, Romina Barria, Marc R Freeman.
      The Sarm1 NAD + hydrolase drives neurodegeneration in many contexts, but how Sarm1 activity is regulated remains poorly defined. Using CRISPR/Cas9 screening, we found loss of VHL suppressed Sarm1-mediated cellular degeneration. VHL normally promotes O 2 -dependent constitutive ubiquitination and degradation of hypoxia-inducible factor 1 (HIF-1), but during hypoxia, HIF-1 is stabilized and regulates gene expression. We observed neuroprotection after depletion of VHL or other factors required for HIF-1 degradation, and expression of a non-ubiquitinated HIF-1 variant led to even stronger blockade of axon degeneration in mammals and Drosophila . Neuroprotection required HIF-1 DNA binding, prolonged expression, and resulted in broad gene expression changes. Unexpectedly, stabilized HIF-1 prevented the precipitous NAD + loss driven by Sarm1 activation in neurons, despite NAD + hydrolase activity being intrinsic to the Sarm1 TIR domain. Our work argues hypoxia inhibits Sarm1 activity through HIF-1 driven transcriptional changes, rendering neurons less sensitive to Sarm1-mediated neurodegeneration when in a hypoxic state.
    Competing interests: Marc Freeman is co-founder of Nura Bio, a biotech startup pursuing novel neuroprotective therapies including SARM1 inhibition. The remaining authors declare no competing interests.
    DOI:  https://doi.org/10.1101/2025.01.17.633664
  58. Nat Commun. 2025 Jan 09. 16(1): 543
    Deacetylated SNAP47 recruits HOPS to facilitate autophagosome-lysosome fusion independent of STX17.
    Fenglei Jian, Shen Wang, Wenmin Tian, Yang Chen, Shixuan Wang, Yan Li, Cong Ma, Yueguang Rong.
      Autophagy, a conserved catabolic process implicated in a diverse array of human diseases, requires efficient fusion between autophagosomes and lysosomes to function effectively. Recently, SNAP47 has been identified as a key component of the dual-purpose SNARE complex mediating autophagosome-lysosome fusion in both bulk and selective autophagy. However, the spatiotemporal regulatory mechanisms of this SNARE complex remain unknown. In this study, we found that SNAP47 undergoes acetylation followed by deacetylation during bulk autophagy and mitophagy. The acetylation status of SNAP47 is regulated by the acetyltransferase CBP and the deacetylase HDAC2. Notably, the spatiotemporal regulatory dynamics of SNAP47 acetylation differ between bulk autophagy and mitophagy due to distinct regulation on the activity of acetyltransferase and deacetylase. Acetylated SNAP47 inhibits autophagosome-lysosome fusion by indirectly impeding SNARE complex assembly. Mechanistically, deacetylated SNAP47 recruits HOPS components to autophagic vacuoles independently of STX17 and STX17-SNAP47 interaction, while acetylated SNAP47 inhibits this recruitment, consequently leading to the failure of SNARE complex assembly. Taken together, our study uncovers a SNAP47 acetylation-dependent regulatory mechanism governing autophagosome-lysosome fusion by modulating the recruitment of HOPS to autophagic vacuoles without involving STX17, SNAP47-STX17 interaction and ternary SNARE complex formation.
    DOI:  https://doi.org/10.1038/s41467-025-55906-x
  59. Nucleic Acids Res. 2025 Jan 24. pii: gkaf036. [Epub ahead of print]53(3):
    The ortholog of human DNAJC9 promotes histone H3-H4 degradation and is counteracted by Asf1 in fission yeast.
    Yan Ding, Jun Li, He-Li Jiang, Fang Suo, Guang-Can Shao, Xiao-Ran Zhang, Meng-Qiu Dong, Chao-Pei Liu, Rui-Ming Xu, Li-Lin Du.
      Mammalian J-domain protein DNAJC9 interacts with histones H3-H4 and is important for cell proliferation. However, its exact function remains unclear. Here, we show that, in the fission yeast Schizosaccharomyces pombe, loss of Djc9, the ortholog of DNAJC9, renders the histone chaperone Asf1 no longer essential for growth. Utilizing AlphaFold-based structural prediction, we identified a histone-binding surface on Djc9 that binds to helix α3 of H3 in a manner that precludes simultaneous helix α3-binding by Asf1. Djc9 and Asf1 indeed compete for binding to the H3-H4 dimer in vitro, and an H3-α3 mutation impeding Djc9 binding also renders Asf1 non-essential, indicating that the role of Asf1 needed for growth in fission yeast is to prevent histone binding by Djc9. In the absence of Asf1, cell growth is hindered due to unrestrained Djc9-mediated downregulation of H3 and H4. In the presence of Asf1, Djc9 confers resistance to the DNA replication inhibitor hydroxyurea and dominant negative disease-related histone mutants by promoting the degradation of superfluous or dysfunctional histones. Our findings provide new insights into the function and mechanism of this conserved histone-binding protein.
    DOI:  https://doi.org/10.1093/nar/gkaf036
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